Protein-induced morphogenesis

ABSTRACT

Disclosed are 1) amino acid sequence data, structural features, homologies and various other data characterizing morphogenic proteins, 2) methods of producing these proteins from natural and recombinant sources and from synthetic constructs, 3) morphogenic devices comprising these morphogenic proteins and a suitably modified tissue-specific matrix, and 4) methods of inducing non-chondrogenic tissue growth in a mammal.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of co-pending U.S.Ser. No. 667,274, filed Mar. 11, 1991.

BACKGROUND OF THE INVENTION

[0002] This invention relates to morphogenic proteins which can inducetissue morphogenesis in mammals; to methods of identifying theseproteins and obtaining them from natural sources or producing syntheticforms of these proteins by expressing recombinant DNA encoding theproteins; to the fabrication of tissue-specific acellular matrices; andto methods for promoting tissue stasis, repair and regeneration, andmethods for increasing progenitor cell populations using these proteins.

[0003] Cell differentiation is the central characteristic ofmorphogenesis which initiates in the embryo, and continues to variousdegrees throughout the life of an organism in adult tissue repair andregeneration mechanisms. The degree of morphogenesis in adult tissuevaries among different tissues and is related, among other things, tothe degree of cell turnover in a given tissue. On this basis, tissuescan be divided into three broad categories: (1) tissues with static cellpopulations such as nerve and skeletal muscle where there is no celldivision and most of the cells formed during early development persistthroughout adult life; (2) tissues containing conditionally renewingpopulations such as liver where there is generally little cell divisionbut, in response to an appropriate stimulus, cells can divide to producedaughters of the same differentially defined type; and (3) tissues withpermanently renewing populations including blood, testes and stratifiedsquamous epithelia which are characterized by rapid and continuous cellturnover in the adult. Here, the terminally differentiated cells have arelatively short life span and are replaced through proliferation of adistinct subpopulation of cells, known as stem or progenitor cells.

[0004] The cellular and molecular events which govern the stimulus fordifferentiation of these cells is an area of intensive research. In themedical field, it is anticipated that the discovery of factor(s) whichcontrol cell differentiation and tissue morphogenesis will significantlyadvance medicine's ability to repair and regenerate diseased or damagedmammalian tissues and organs. Particularly useful areas includereconstructive surgery and in the treatment of tissue degenerativediseases including arthritis, emphysema, osteoporosis, cardiomyopathy,cirrhosis, and degenerative nerve diseases.

[0005] A number of different factors have been isolated in recent yearswhich appear to play a role in cell differentiation. Some of thesefactors are gene transcription activators such as the NOTCH gene,identified in Drosophila and the related XOTCH gene identified inXenopus, as well as a number of transcription activators identified inCaenorhabditis elegans.

[0006] The hemopoietic system, because of its continually renewing cellpopulation, is an area of concentrated study. Factors identified in thissystem which may be involved in cell renewal include interleukin 3(IL-3), erythropoietin, the CSFs (GM-CSF, G-CSF, M-CSF et al.) andvarious stem cell growth factors.

[0007] Other proteins thought to play a role in cell differentiationinclude proteins that are members of the family of insulin-like growthfactors (IGF), members of the family of heparin-binding growth factors,(e.g., FGF—acidic and basic fibroblast growth factors, andECDGF—embryonal carcinoma-derived growth factor) as well as severaltransforming oncogenes (hst and int-2, see for example, Heath et al.,(1988), J. Cell Sci. Suppl. 10:256-256.) DIF (Differentiation InducingFactor), identified in Dictyostelium discoideum, is anotherbioregulatory protein, directing prestock cell differentiation in thatorganism.

[0008] The structurally related proteins of the TGF-β superfamily ofproteins also have been identified as involved in a variety ofdevelopmental events. For example, TGF-β and the polypeptides of theinhibin/activin group appear to play a role in the regulation of cellgrowth and differentiation. MIS (Mullerian Inhibiting Substance) causesregression of the Mullerian duct in development of the mammalian maleembryo, and DPP, the gene product of the Drosophila decapentaplegiccomplex is required for appropriate dorsal-ventral specification.Similarly, Vg-1 is involved in mesoderm induction in Xenopus, and Vgr-1has been identified in a variety of developing murine tissues.

[0009] Another source that has revealed a wealth of information is inthe area of bone morphogenesis. The development and study of a bonemodel system has identified the developmental cascade of bonedifferentiation as consisting of chemotaxis of mesenchymal cells,proliferation of these progenitor cells, differentiation of these cellsinto of cartilage, vascular invasion, bone formation, remodeling, andfinally, marrow differentiation (Reddi (1981) Collagen Rel. Res.1:209-206). Proteins capable of inducing endochondral bone formation ina mammal when implanted in association with a matrix now have beenidentified in a number of different mammalian species, as have the genesencoding these proteins, (see, for example, U.S. Pat. No. 4,968,590;U.S. Ser. No. 315,342 filed Feb. 23, 1989; and U.S. Ser. No. 599,543,filed Oct. 18, 1990). These proteins, which share significant amino acidsequence homology with one another as well as structural similaritieswith various members of the TGF-β super family of proteins, have beenshown to induce endochondral bone formation and/or bone cartilageformation when implanted in a mammal in association with a suitablymodified matrix. Proteins capable of inducing a similar developmentalcascade of tissue morphogenesis of other tissues have not beenidentified.

[0010] It is an object of this invention to provide morphogenic proteins(“morphogens”), and methods for identifying these proteins, which arecapable of inducing the developmental cascade of tissue morphogenesisfor a variety of tissues in mammals different from bone or bonecartilage. This morphogenic activity includes the ability to induceproliferation and differentiation of progenitor cells, and the abilityto support and maintain the differentiated phenotype through theprogression of events that results in the formation of adult tissue.Another object is to provide genes encoding these proteins as well asmethods for the expression and isolation of these proteins, from eithernatural sources or biosynthetic sources, using recombinant DNAtechniques. Still another object is to provide tissue-specific acellularmatrices that may be used in combination with these proteins, andmethods for their production. Other objects include providing methodsfor increasing a progenitor cell population in a mammal, methods forstimulating progenitor cells to differentiate in vivo or in vitro andmaintain their differentiated phenotype, methods for inducingtissue-specific growth in vivo and methods for the replacement ofdiseased or damaged tissue in vivo. These and other objects and featuresof the invention will be apparent from the description, drawings, andclaims which follow.

SUMMARY OF THE INVENTION

[0011] This invention provides morphogenic proteins (“morphogens”)capable of inducing the developmental cascade of tissue morphogenesis ina mammal. In particular, these proteins are capable of inducing theproliferation of uncommitted progenitor cells, and inducing thedifferentiation of these stimulated progenitor cells in atissue-specific manner under appropriate environmental conditions. Inaddition, the morphogens are capable of supporting the growth andmaintenance of these differentiated cells. These morphogenic activitiesallow the proteins of this invention to initiate and maintain thedevelopmental cascade of tissue morphogenesis in an appropriate,morphogenically permissive environment, stimulating stem cells toproliferate and differentiate in a tissue-specific manner, and inducingthe progression of events that culminate in new tissue formation. Thesemorphogenic activities also allow the proteins to stimulate the“redifferentiation” of cells previously induced to stray from theirdifferentiation path. Under appropriate environmental conditions it isanticipated that these morphogens also may stimulate the“dedifferentiation” of committed cells (see infra.)

[0012] In one aspect of the invention, the proteins and compositions ofthis invention are useful in the replacement of diseased or damagedtissue in a mammal, particularly when the damaged tissue interferes withnormal tissue or organ function. Accordingly, it is anticipated that theproteins of this invention will be useful in the repair of damagedtissue such as, for example, damaged lung tissue resulting fromemphysema, cirrhotic kidney or liver tissue, damaged heart or bloodvessel tissue, as may result from cardiomyopathies and/oratherothrombotic or cardioembolic strokes, damaged stomach tissueresulting from ulceric perforations or their repair, damaged neuraltissue as may result from physical injury, degenerative diseases such asAlzheimer's disease or multiple sclerosis or strokes, damaged dentintissue as may result from disease or mechanical injury, and damagedcartilage and ligament tissue. When the proteins of this invention areprovided to, or their expression stimulated at, a tissue-specific locus,the developmental cascade of tissue morphogenesis is induced (seeinfra). Cells stimulated ex vivo by contact with the proteins or agentscapable of stimulating morphogen expression in these cells also may beprovided to the tissue locus. In these cases the existing tissueprovides the necessary matrix requirements, providing a suitablesubstratum for the proliferating and differentiating cells in amorphogenically permissive environment, as well as providing thenecessary signals for directing the tissue-specificity of the developingtissue. Alternatively, the proteins or stimulated cells may be combinedwith a formulated matrix and implanted as a device at a locus in vivo.The formulated matrix should be a biocompatible, preferablybiodegradable, appropriately modified tissue-specific acellular matrixhaving the characteristics described below.

[0013] In many instances, the loss of tissue function results from scartissue, formed in response to an initial or repeated injury to thetissue. The degree of scar tissue formation generally depends on theregenerative properties of the injured tissue, and on the degree andtype of injury. Thus, in another aspect, the invention includesmorphogens that may be used to prevent or substantially inhibit theformation of scar tissue by providing the morphogens, ormorphogen-stimulated cells, to a newly injured tissue loci (see infra).

[0014] The morphogens of this invention also may be used to increase orregenerate a progenitor or stem cell population in a mammal. Forexample, progenitor cells may be isolated from an individual's bonemarrow, stimulated ex vivo for a time and at a morphogen concentrationsufficient to induce the cells to proliferate, and returned to the bonemarrow. Other sources of progenitor cells that may be suitable includebiocompatible cells obtained from a cultured cell line, stimulated inculture, and subsequently provided to the body. Alternatively, themorphogen may be provided systemically, or implanted, injected orotherwise provided to a progenitor cell population in an individual toinduce its mitogenic activity in vivo. For example, an agent capable ofstimulating morphogen expression in the progenitor cell population ofinterest may be provided to the cells in vivo, for example systemically,to induce mitogenic activity. Similarly, a particular population ofhemopoietic stem cells may be increased by the morphogens of thisinvention, for example by perfusing an individual's blood to extract thecells of interest, stimulating these cells ex vivo, and returning thestimulated cells to the blood. It is anticipated that the ability toaugment an individual's progenitor cell population will significantlyenhance existing methods for treating disorders resulting from a loss orreduction of a renewable cell population. Two particularly significantapplications include the treatment of blood disorders and impairment orloss of immune function. Other cell populations whose proliferation maybe exploited include the stem cells of the epidermis, which may be usedin skin tissue regeneration, and the stem cells of the gastrointestinallining for healing of ulcers.

[0015] In still another aspect of the invention, the morphogens also maybe used to support the growth and maintenance of differentiated cells,inducing existing differentiated cells to continue expressing theirphenotype. It is anticipated that this activity will be particularlyuseful in the treatment of tissue disorders where loss of function iscaused by cells becoming senescent or quiescent, such as may occur inosteoporosis. Application of the protein directly to the cells to betreated, or providing it by systemic injection, can be used to stimulatethese cells to continue expressing their phenotype, therebysignificantly reversing the effects of the dysfunction (see infra).Alternatively, administration of an agent capable of stimulatingmorphogen expression in vivo also may be used. In addition, themorphogens of this invention also may be used in gene therapy protocolsto stimulate the growth of quiescent cells, thereby potentiallyenhancing the ability of these cells to incorporate exogenous DNA.

[0016] In yet another aspect of the invention, the morphogens of thisinvention also may be used to induce “redifferentiation” of cells thathave strayed from their differentiation pathway, such as can occurduring tumorgenesis. It is anticipated that this activity of theproteins will be particularly useful in treatments to reduce orsubstantially inhibit the growth of neoplasms. The method also isanticipated to induce the de-and re-differentiation of these cells. Asdescribed supra, the proteins may be provided to the cells directly orsystemically, or an agent capable of stimulating morphogen expression invivo may be provided.

[0017] Finally, modulations of endogenous morphogen levels may bemonitored as part of a method for detecting tissue dysfunction.Specifically, modulations in endogenous morphogen levels are anticipatedto reflect changes in tissue or organ stasis, and can be followed bymonitoring fluctuations in the body's natural antibody titer tomorphogens.

[0018] The morphogenic proteins and compositions of this invention canbe isolated from a variety of naturally-occurring sources, or they maybe constructed biosynthetically using conventional recombinant DNAtechnology. Similarly, the matrices may be derived from organ-specifictissue, or they may be formulated synthetically, as described below.

[0019] A key to these developments was the discovery andcharacterization of naturally-occurring osteogenic proteins followed byobservation of their remarkable properties. These proteins, originallyisolated from bone, are capable of inducing the full developmentalcascade of bone formation, including vascularization, mineralization,and bone marrow differentiation, when implanted in a mammalian body inassociation with a suitably modified matrix. Native proteins capable ofinducing this developmental cascade, as well as DNA sequences encodingthese proteins now have been isolated and characterized for a number ofdifferent species (e.g., OP-1, OP-2, and CBMP-2. See, for example, U.S.Pat. Nos. 4,968,590 and 5,011,691; U.S. application Ser. No. 422,699,filed Oct. 17, 1989; and U.S. Ser. No. 600,024 and 599,543, both filedOct. 18, 1990; Sampath et al. (1990) J. Bio. Chem 265:13198-13205 andOzkaynak, et al. (1990) EMBO 9:2085-2093). The mature forms of theseproteins share substantial amino acid sequence homology, especially inthe C-terminal regions of the mature proteins. In particular, theproteins share a conserved six or seven cysteine skeleton in this region(e.g., the linear arrangement of these C-terminal cysteine residues isessentially conserved in the different proteins, in addition to other,apparently required amino acids (see Table II, infra).

[0020] Polypeptide chains not normally associated with bone or boneformation, but sharing substantial amino acid sequence homology with theC-terminus of the osteogenic proteins, including the conserved six orseven cysteine skeleton, also have been identified as competent forinducing bone in mammals. Among these are amino acid sequencesidentified in Drosophila and Xenopus, (e.g., DPP and Vgl; see, forexample, U.S. Pat. No. 5,011,691 and Table II, infra). In addition,non-native biosynthetic constructs designed based on extrapolation fromthese sequence homologies, including the conserved six or seven cysteineskeleton, have been shown to induce endochondral bone formation inmammals when implanted in association with an appropriate matrix (SeeTable III, infra).

[0021] It has now been discovered that this “family” of proteins sharingsubstantial amino acid sequence homology and the conserved six or sevencysteine skeleton are true morphogens, capable of inducing, in additionto bone and bone cartilage, tissue-specific morphogenesis for a varietyof other organs and tissues. The proteins apparently bind to surfacereceptors or otherwise contact and interact with progenitor cells,predisposing or stimulating the cells to proliferate and differentiatein a morphogenically permissive environment. The morphogens are capableof inducing the developmental cascade of cellular and molecular eventsthat culminate in the formation of new organ-specific tissue, includingany vascularization, connective tissue formation, and nerve ennervationas required by the naturally occurring tissue.

[0022] It also has been discovered that the way in which the cellsdifferentiate, whether, for example, they differentiate intobone-producing osteoblasts, hemopoietic cells, or liver cells, dependson the nature of their local environment (see infra). Thus, in additionto requiring a suitable substratum on which to anchor, the proliferatingand differentiating cells also require appropriate signals to directtheir tissue-specificity. These signals may take the form of cellsurface markers. Thus, in a suitable, typically bone powder-derivedmatrix presented in a vascular supported environment, themorphogen-activated progenitor cells differentiate not only through thebone-producing cascade including transformation to chondrocytes and thento osteoblasts, including formation of the necessary associated vascularnetwork.

[0023] When the morphogens (or progenitor cells stimulated by thesemorphogens) are provided at a tissue-specific locus (e.g., by systemicinjection or by implantation or injection at a tissue-specific locus, orby administration of an agent capable of stimulating morphogenexpression in vivo), the existing tissue at that locus, whether diseasedor damaged, has the capacity of acting as a suitable matrix.Alternatively, a formulated matrix may be externally provided togetherwith the stimulated progenitor cells or morphogen, as may be necessarywhen the extent of injury sustained by the damaged tissue is large. Thematrix should be a biocompatible, suitably modified acellular matrixhaving dimensions such that it allows the influx, differentiation, andproliferation of migratory progenitor cells, and is capable of providinga morphogenically permissive environment (see infra). The matrixpreferably is tissue-specific, and biodegradable.

[0024] Formulated matrices may be generated from dehydratedorgan-specific tissue, prepared for example, by treating the tissue withsolvents to substantially remove the non-structural components from thetissue. Alternatively, the matrix may be formulated synthetically usinga biocompatible, preferably in vivo biodegradable, structural polymersuch as collagen in association with suitable tissue-specific cellattachment factors. Currently preferred structural polymers comprisetissue-specific collagens. Currently preferred cell attachment factorsinclude glycosaminoglycans and proteoglycans. The matrix further may betreated with an agent or agents to increase the number of pores andmicropits on its surfaces, so as to enhance the influx, proliferationand differentiation of migratory progenitor cells from the body of themammal.

[0025] Among the proteins useful in this invention are proteinsoriginally identified as osteogenic proteins, such as the OP-1, OP-2 andCBMP2 proteins, as well as amino acid sequence related proteins such asDPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse, see TableII and Seq. ID Nos.5-14), and the recently identified GDF-1 protein(Seq. ID No. 14). The members of this family, which include members ofthe TGF-β super-family of proteins, share substantial amino acidsequence homology in their C-terminal regions. Table I, below, describesthe various morphogens identified to date, including their nomenclatureas used herein, and Seq. ID references. TABLE I “OP-1” refersgenerically to the group of active proteins expressed from part or allof a DNA sequence encoding OP-1 protein, e.g., human OP-1 (“hOP-1”, Seq.ID No. 5, mature protein amino acid sequence), or mouse OP-1 (“mOP-1”,Seq. ID No. 6, mature protein amino acid sequence.) The conserved sevencysteine skeleton is defined by residues 38 to 139 of Seq. ID Nos. 5 and6. “OP-2” refers generically to the group of active proteins expressedfrom part or all of a DNA sequence encoding OP-2 protein, e.g., humanOP-2 (“hOP-2”, Seq. ID No. 7, mature protein amino acid sequence) ormouse OP-2 (“mOP-2”, Seq. ID No. 8, mature protein amino acid sequence).The conserved seven cysteine sekelton is defined by residues 38 to 139of Seq. ID Nos. 7 and 8. “CBMP2” refers generically to the activeproteins expressed from a DNA sequence encoding CBMP2 protein, e.g.,human CBMP2 (“CBMP2B(fx)”, Seq ID No. 9) or bovine CBMP2 DNA(“CBMP2A(fx)”, Seq. ID No. 10). “Vgl(fx)” refers to protein sequencesencoded by the xenopus Vgl gene and defining the conserved sevencysteine skeleton (Seq. ID No. 11). “Vgr-1(fx)” refers to proteinsequences encoded by the murine Vgr-1 gene and defining the conservedseven cysteine skeleton (Seq. ID No. 12). “DPP(fx)” refers to proteinsequences encoded by the Drosophila DPP gene and defining the conservedseven cysteine skeleton (seq. ID No. 13). “GDF-1(fx)” refers to proteinsequences encoded by the human GDF-1 gene and defining the conservedseven cysteine skeleton (seq. ID No. 14).

[0026] The OP-2 proteins have an additional cysteine residue in thisregion (position 41), in addition to the conserved cysteine skeleton incommon with the other proteins in this family. The GDF-1 protein has afour amino acid insert within the conserved skeleton (residues 44-47 ofSeq. ID No. 14) but this insert likely does not interfere with therelationship of the cysteines in the folded structure. In addition, theCBMP2 proteins are missing one amino acid residue within the cysteineskeleton.

[0027] The morphogens are inactive when reduced, but are active asoxidized homodimers and as various oxidized heterodimers. Thus, asdefined herein, a morphogen of this invention is a dimeric proteincomprising a pair of polypeptide chains, wherein each polypeptide chaincomprises at least the C-terminal six cysteine skeleton defined byresidues 43-139 of Seq. ID No. 5, including functionally equivalentarrangements of these cysteines (e.g., amino acid insertions ordeletions which alter the linear arrangement of the cysteines in thesequence but not their relationship in the folded structure), such that,when the polypeptide chains are folded, the dimeric protein speciescomprising the pair of polypeptide chains has the appropriatethree-dimensional structure, including the appropriate intra- orinter-chain disulfide bonds such that the protein is capable of actingas a morphogen as defined herein. Specifically, the protein is capableof any of the following biological functions in a morphogenicallypermissive environment: stimulating proliferation of progenitor cells;stimulating the differentiation of progenitor cells; stimulating theproliferation of differentiated cells; and supporting the growth andmaintenance of differentiated cells, including the “redifferentiation”of these cells. In addition, it is also anticipated that the morphogensof this invention will be capable of inducing dedifferentiation ofcommitted cells under appropriate environmental conditions.

[0028] In one preferred aspect, the morphogens of this inventioncomprise one of two species of generic amino acid sequences: GenericSequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID No. 2); whereeach Xaa indicates one of the 20 naturally-occurring L-isomer, α-aminoacids or a derivative thereof. Generic Sequence 1 comprises theconserved six cysteine skeleton and Generic Sequence 2 comprises theconserved six cysteine skeleton plus the additional cysteine identifiedin OP-2. In another preferred aspect, these sequences further comprisethe following sequence at their N-terminus: Cys Xaa Xaa Xaa Xaa   1               5

[0029] Preferred amino acid sequences within the foregoing genericsequences include: Generic Sequence 3 (Seq. ID No. 3) and GenericSequence 4 (Seq. ID No. 4), listed below, which accommodate thehomologies shared among the various members of this morphogen familyidentified to date, as well as the amino acid sequence variation amongthem. Note that these generic sequences allow for an additional cysteineat position 41 or 46 in Generic Sequences 3 or 4, respectively,providing an appropriate cysteine skeleton where interor intramoleculardisulfide bonds can form, and contain certain critical amino acids whichinfluence the tertiary structure of the proteins. Generic Sequence 3    Leu Tyr Val Xaa Phe     1                5 Xaa Xaa Xaa Gly Trp XaaXaa Trp Xaa                  10 Xaa Ala Pro Gly Xaa Xaa Xaa Ala  15                 20 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa         25                 30 Xaa Pro Xaa Xaa Xaa Xaa Xaa                  35 XaaXaa Xaa Asn His Ala Xaa Xaa         40                  45 Xaa Xaa LeuXaa Xaa Xaa Xaa Xaa                  50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys     55                  60 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa             65 Xaa Xaa Xaa Leu Xaa Xaa Xaa  70                  75 XaaXaa Xaa Xaa Val Xaa Leu Xaa              80 Xaa Xaa Xaa Xaa Met Xaa ValXaa  85                  90 Xaa Cys Gly Cys Xaa          95

[0030] wherein each Xaa is independently selected from a group of one ormore specified amino acids defined as follows: “Res.” means “residue”and Xaa at res.4=(Ser, Arg, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser orLys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or Val); Xaa atres.11=(Gln, Leu, Asp, His or Asn); Xaa at res.12=(Asp, Arg or Asn); Xaaat res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.18=(Glu,Gln, Leu, Lys, Pro or Arg); Xaa at res.20=(Tyr or Phe); Xaa atres.21=(Ala, Ser, Asp, Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn orPhe); Xaa at res.26=(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu,Lys, Asp or Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa atres.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu or Val); Xaa atres.34=(Asn, Asp, Ala or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu orAla); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met,Phe, Gly or Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser orGly); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaaat res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr,Ala or Ser); Xaa at res.49=(Val or Met); Xaa at res.50=(His or Asn); Xaaat res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met,Asn, Ala or Val); Xaa at res.53=(Asn, Lys, Ala or Glu); Xaa atres.54=(Pro or Ser); Xaa at res.55=(Glu, Asp, Asn, or Gly); Xaa atres.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.57=(Val,Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys or Leu); Xaaat res.60=(Pro or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thror Ala); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Metor Val); Xaa at res.68=(Asn, Ser or Asp); Xaa at res.69=(Ala, Pro orSer); Xaa at res.70=(Ile, Thr or Val); Xaa at res.71=(Ser or Ala); Xaaat res.72=(Val or Met); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe,Tyr or Leu); Xaa at res.76=(Asp or Asn); Xaa at res.77=(Asp, Glu, Asn orSer); Xaa at res.78=(Ser, Gln, Asn or Tyr); Xaa at res.79=(Ser, Asn, Aspor Glu); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile or Val);Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln or His); Xaa atres.86=(Tyr, Ala or His); Xaa at res.87=(Arg, Gln or Glu); Xaa atres.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr or Ala); Xaa atres.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly or Glu); andXaa at res.97=(His or Arg); and Generic Seq. 4: Generic Sequence 4 CysXaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe  1                5                 10 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa                 15 Xaa Ala Pro Xaa Gly Xaa Xaa Ala  20                 25 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa          30                 35 Xaa Pro Xaa Xaa Xaa Xaa Xaa                  40 AsnXaa Xaa Asn His Ala Xaa Xaa          45                  50 Xaa Xaa LeuXaa Xaa Xaa Xaa Xaa                  55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys     60                  65 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa             70 Xaa Xaa Xaa Leu Xaa Xaa Xaa  75                  80 XaaXaa Xaa Xaa Val Xaa Leu Xaa              85 Xaa Xaa Xaa Xaa Met Xaa ValXaa  90                  95 Xaa Cys Gly Cys Xaa         100

[0031] wherein each Xaa is independently selected from a group of one ormore specified amino acids as defined by the following: “Res.” means“residue” and Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or Arg); Xaaat res.4=(His or Arg); Xaa at res.5=(Glu, Ser, His, Gly, Arg or Pro);Xaa at res.9=(Ser, Arg, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser orLys); Xaa at res.12=(Asp or Glu); Xaa at res.13=(Leu or Val); Xaa atres.16 (Gln, Leu, Asp, His or Asn); Xaa at res.17=(Asp, Arg, or Asn);Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or Val); Xaa atres.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.25=(Tyr or Phe); Xaaat res.26=(Ala, Ser, Asp, Met, His, Leu, or Gln); Xaa at res.28=(Tyr,Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp or Gln); Xaa atres.33=Glu, Lys, Asp or Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa atres.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu or Val); Xaa atres.39=(Asn, Asp, Ala or Thr); Xaa at res.40=(Ser, Asp, Glu, Leu orAla); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met,Phe, Gly or Leu); Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr,Leu or Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaaat res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa atres.54=(Val or Met); Xaa at res.55=(His or Asn); Xaa at res.56=(Phe,Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala or Val);Xaa at res.58=(Asn, Lys, Ala or Glu); Xaa at res.59=(Pro or Ser); Xaa atres.60=(Glu, Asp, or Gly); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr,Ser or Ala); Xaa at res.62=(Val, Ala or Ile); Xaa at res.63=(Pro orAsp); Xaa at res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa atres.68=(Ala or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln,Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn,Ser or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr orVal); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met); Xaa atres.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at res.81=(Aspor Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser, Gln,Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or Glu); Xaa at res.85=(Asn,Thr or Lys); Xaa at res.87=(Ile or Val); Xaa at res.89=(Lys or Arg); Xaaat res.90=(Lys, Asn, Gln or His); Xaa at res.91=(Tyr, Ala or His); Xaaat res.92=(Arg, Gln or Glu); Xaa at res.93=(Asn, Glu or Asp); Xaa atres.95=(Val, Thr or Ala); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaaat res.98=(Ala, Gly or Glu); and Xaa at res.102=(His or Arg).

[0032] Particularly useful sequences include the C-terminal residues ofVgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B and GDF-1 (see Table II,infra, and Seq. ID Nos. 5-12) which include at least the conserved sixor seven cysteine skeleton. In addition, biosynthetic constructsdesigned from the generic sequences, such as COP-1, 3-5, 7, 16 (seeTable III, infra) also are useful. Others include CBMP3 and theinhibins/activin proteins. Accordingly, other useful sequences are thosesharing at least 70% amino acid sequence homology, and preferably 80%homology with any of the sequences above. These are anticipated toinclude allelic and species variants and mutants, and biosyntheticmuteins, as well as novel members of this morphogenic family ofproteins.

[0033] The invention thus provides proteins comprising any of thepolypeptide chains described above, whether isolated fromnaturally-occurring sources, or produced by recombinant DNA techniques,and includes allelic and species variants of these proteins,naturally-occurring or biosynthetic mutants thereof, as well as varioustruncated and fusion constructs. Deletion or addition mutants also areenvisioned to be active (see infra), including those which may alter theconserved C-terminal cysteine skeleton, provided that the alterationdoes not functionally disrupt the relationship of these cysteines in thefolded structure. Accordingly, such active forms are considered theequivalent of the specifically described constructs disclosed herein.The proteins may include forms having varying glycosylation patterns,varying N-termini, a family of related proteins having regions of aminoacid sequence homology, and active truncated or mutated forms of nativeor biosynthetic proteins, produced by expression of recombinant DNA inhost cells.

[0034] The morphogenic proteins can be expressed from intact ortruncated cDNA or from synthetic DNAs in procaryotic or eucaryotic hostcells, and purified, cleaved, refolded, and dimerized to formmorphogenically active compositions. Currently preferred host cellsinclude E. coli or mammalian cells, such as CHO, COS or BSC cells.

[0035] Thus, in view of this disclosure, skilled genetic engineers canisolate genes from cDNA or genomic libraries of various differentspecies-which encode appropriate amino acid sequences, or construct DNAsfrom oligonucleotides, and then can express them in various types ofhost cells, including both procaryotes and eucaryotes, to produce largequantities of active proteins capable of inducing tissue-specific celldifferentiation and tissue morphogenesis in mammals including humans.

[0036] The invention thus further comprises these methods of inducingtissue-specific morphogenesis using the morphogenic proteins of thisinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The foregoing and other objects and features of this invention,as well as the invention itself, may be more fully understood from thefollowing description, when read together with the accompanyingdrawings, in which:

[0038]FIG. 1 is a photomicrograph of a Northern Blot identifying Vgr-1specific transcripts in various adult murine tissues;

[0039]FIG. 2 is a photomicrograph of a Northern Blot identifyingmOP-1-specific mRNA expression in various murine tissues prepared from 2week old mice (panel A) and 5 week old mice (Panel B);

[0040]FIG. 3 is a photomicrograph of Northern Blots identifying mRNAexpression of EF-Tu (A, control), mOP-1 (B, D), and Vgr-1 (C) in (1)17-day embryos and (2) 3-day post natal mice;

[0041]FIGS. 4A and 4B are photomicrographs showing the presence of OP-1(by immunofluorescence staining) in the cerebral cortex (A) and spinalcord (B);

[0042]FIGS. 5A and 5B are photomicrographs illustrating the ability ofmorphogen (OP-1) to induce undifferentiated NG108 calls (5A) to undergodifferentiation of neural morphology (5B).

[0043] FIGS. 6A-6D are photomicrographs showing the effect of morphogen(OP-1) on human embry carcinoma cell redifferentiation;

[0044]FIG. 7 is a photomicrograph showing the effects of phosphatebuffered saline (PBS, animal 1) or morphogen (OP-1, animal 2) onpartially hepatectomized rats;

[0045] FIGS. 8A-8C are photomicrographs showing the effect of notreatment (8A), carrier matrix treatment (8B) and morphogen treatment(OP-1,8C) on dentin regeneration.

DETAILED DESCRIPTION

[0046] Purification protocols first were developed which enabledisolation of the osteogenic (bone inductive) protein present in crudeprotein extracts from mammalian bone. (See PCT US 89/01453, and U.S.Pat. No. 4,968,590.) The development of the procedure, coupled with theavailability of fresh calf bone, enabled isolation of substantially purebovine osteogenic protein (BOP). BOP was characterized significantly;its ability to induce bone cartilage and ultimately endochondral bonegrowth in cat, rabbit, and rat were demonstrated and studied; it wasshown to be able to induce the full developmental cascade of boneformation previously ascribed to unknown protein or proteins inheterogeneous bone extracts. This dose dependent and highly specificactivity was present whether or not the protein was glycosylated (seeU.S. Pat. No. 4,968,958, filed Apr. 8, 1988 and Sampath et al., (1990)J. Biol. Chem. 265: pp. 13198-13205). Sequence data obtained from thebovine materials suggested probe designs which were used to isolategenes encoding osteogenic proteins from different species. Human andmurine OP counterparts have now been identified and characterized (see,for example, U.S. Ser. No. 422,699, filed Oct. 17, 1989 and disclosingDNA and amino acid sequence for human OP-1 (“hOP-1”); U.S. Ser. No.600,024 filed Oct. 18, 1990, disclosing the murine OP-1 DNA and encodedamino acid sequence (“mOP-1”) and U.S. Ser. No. 599,543, filed Oct. 18,1990), disclosing the human and murine DNA and amino acid sequences forOP-2 (“hOP-2” and mOP-2”.)

[0047] Sequence data from the bovine materials also suggestedsubstantial homology with a number of proteins known in the art whichwere not known to play a role in bone formation. Bone formation assaysperformed with these proteins showed that, when these proteins wereimplanted in a mammal in association with a suitable matrix, cartilageand endochondral bone formation was induced. (see, for example, U.S.Pat. No. 5,011,691.) One of these proteins is DPP, a Drosophila proteinknown to play a role in dorsal-ventral specification and required forthe correct morphogenesis of the imaginal discs. Two other proteins arerelated sequences identified in Xenopus and mouse (Vgl and Vgr-1,respectively), thought to play a role in the control of growth anddifferentiation during embryogenesis. While DPP and Vgr-1 (orVgr-1-like) transcripts have been identified in a variety of tissues(embryonic, neonatal and adult, Lyons et al., (1989) PNAS 86:4554-4558,and see infra), Vgl transcripts, which are maternally inherited andspacially restricted to the vegetal endoderm, decline dramatically aftergastrulation.

[0048] From these homologies a generic consensus sequence was derivedwhich encompasses the minimally required active sequence for inducingbone morphogenesis in a mammal when implanted in association with amatrix. The generic sequence has at least a conserved six cysteineskeleton (Generic Sequence 1, Seq. ID No. 1) or, optionally, a7-cysteine skeleton (Generic Sequence 2, Seq. ID No. 2), where each Xaaindicates any one of the 20 naturally-occurring L-isomer, α-amino acidsor a derivative thereof. Longer generic sequences which also are usefulfurther comprise the following sequence at their N-termini: Cys Xaa XaaXaa Xaa   1                5

[0049] Biosynthetic constructs designed from this generic consensussequence also have been shown to induce endochondral bone formation(e.g., COP-1, COP-3, COP-4, COP-5, COP-7 and COP-16, see, for example,U.S. Pat. No. 5,011,691. Table II, set forth below, compares the aminoacid sequences of an osteogenically active region of native matureproteins that have been identified as morphogens, including human OP-1(hOP-1, Seq. ID Nos. 7 and 14), mouse OP-1 (mOP-1, Seq. ID No. 15),human and mouse OP-2 (Seq. ID Nos. 8, 16 and 17), CBMP2a (Seq. ID Nos. 9and 8), CBMP2b (Seq. ID Nos. 10 and 29), DPP (from Drosophila, Seq. IDNo. 11), Vgl, (from Xenopus, Seq. ID No. 12), and Vgr (from mouse, Seq.ID No. 13). In the table, three dots indicates that the amino acid inthat position is the same as the amino acid in hOP-1. Three dashesindicates that no amino acid is present in that position, and areincluded for purposes of illustrating homologies. For example, aminoacid residue 60 of CBMP-2A and CBMP-2B is “missing”. Of course, boththese amino acid sequences in this region comprise Asn-Ser (residues 58,59), with CBMP-2A then comprising Lys and Ile, whereas CBMP-2B comprisesSer and Ile. TABLE II hOP-1 Cys Lys Lys His Glu Leu Tyr Val mOP-1 ...... ... ... ... ... ... ... hOP-2 ... Arg Arg ... ... ... ... ... mOP-2... Arg Arg ... ... ... ... ... DPP ... Arg Arg ... Ser ... ... ... Vg1... ... Arg ... His ... ... ... Vgr-1 ... ... ... ... Gly ... ... ...CBMP-2A ... ... Arg ... Pro ... ... ... CBMP-2B ... Arg Arg ... Ser ...... ... GDF-1 ... Arg Ala Arg Arg ... ... ...  1   5 hOP-1 Ser Phe ArgAsp Leu Gly Trp Gln Asp mOP-1 ... ... ... ... ... ... ... ... hOP-2 ...... Gln ... ... ... ... Leu ... mOP-2 Arg ... ... ... ... ... ... Leu... DPP Asp ... Ser ... Val ... ... Asp ... Vg1 Glu ... Lys ... Val ...... ... Asn Vgr-1 ... ... Gln ... Val ... ... ... ... CBMP-2A Asp ...Ser ... Val ... ... Asn ... CBMP-2B Asp ... Ser ... Val ... ... Asn ...GDF-1 ... ... ... Glu Val ... ... His Arg  10  15 hOP-1 Trp Ile Ile AlaPro Glu Gly Tyr Ala mOP-1 ... ... ... ... ... ... ... ... hOP-2 ... Val... ... ... Gln ... ... Ser mOP-2 ... Val ... ... ... Gln ... ... SerDPP ... Val ... ... ... Leu ... ... Asp Vg1 ... Val ... ... ... Gln ...... Met Vgr-1 ... ... ... ... ... Lys ... ... ... CBMP-2A ... ... Val... ... Pro ... ... His CBMP-2B ... ... Val ... ... Pro ... ... GlnGDF-1 ... Val ... ... ... Arg ... Phe Leu  20  25 hOP-1 Ala Tyr Tyr CysGlu Gly Glu Cys Ala mOP-1 ... ... ... ... ... ... ... ... ... [AhOP-2... ... ... ... ... ... ... ... Ser mOP-2 ... ... ... ... ... ... ...... ... DPP ... ... ... ... His ... Lys ... Pro Vg1 ... Asn ... ... Tyr... ... ... Pro Vgr-1 ... Asn ... ... Asp ... ... ... Ser CBMP-2A ...Phe ... ... His ... Glu ... Pro CBMP-2B ... Phe ... ... His ... Asp ...Pro GDF-1 ... Asn ... ... Gln ... Gln ... ...  30  35 hOP-1 Phe Pro LeuAsn Ser Tyr Met Asn Ala mOP-1 ... ... ... ... ... ... ... ... ... hOP-2... ... ... Asp ... Cys ... ... ... mOP-2 ... ... ... Asp ... Cys ...... ... DPP ... ... ... Ala Asp His Phe ... Ser Vg1 Tyr ... ... Thr GluIle Leu ... Gly Vgr-1 ... ... ... ... ... Ala His ... ... ... CBMP-2A... ... ... Ala Asp His Leu ... Ser CBMP-2B ... ... ... Ala Asp His Leu... Ser GDF-1 Leu ... Val Ala Leu Ser Gly Ser** ...  40 hOP-1 Thr AsnHis Ala Ile Val Gln Thr Leu mOP-1 ... ... ... ... ... ... ... ... hOP-2... ... ... ... ... Leu ... Ser ... mOP-2 ... ... ... ... ... Leu ...Ser ... DPP ... ... ... ... Val ... ... ... ... Vg1 Ser ... ... ... ...... ... ... ... Vgr-1 ... ... ... ... ... ... ... ... ... CBMP-2A ...... ... ... ... ... ... ... ... CBMP-2B ... ... ... ... ... ... ... ...... GDF-1 Leu ... ... ... Val Leu Arg Ala ...  45  50 hOP-1 Val His PheIle Asn Pro Glu Thr Val mOP-1 ... ... ... ... ... ... Asp ... ... hOP-2... His Leu Met Lys ... Asn Ala ... mOP-2 ... His Leu Met Lys ... AspVal ... DPP ... Asn ASn Asn ... ... Gly Lys ... Vg1 ... ... Ser ... Glu... ... Asp Ile Vgr-1 ... ... Val Met ... ... ... Tyr ... CBMP-2A ...Asn Ser Val ... Ser --- Lys Ile CBMP-2B ... Asn Ser Val ... Ser --- SerIle GDF-1 Met ... Ala Ala Ala ... Gly Ala Ala  55  60 hOP-1 Pro Lys ProCys Cys Ala Pro Thr Gln mOP-1 ... ... ... ... ... ... ... ... ... hOP-2... ... Ala ... ... ... ... ... Lys mOP-2 ... ... Ala ... ... ... ...... Lys DPP ... ... Ala ... ... Val ... ... ... Vg1 ... Leu ... ... ...Val ... ... Lys Vgr-1 ... ... ... ... ... ... ... ... Lys CBMP-2A ...... Ala ... ... Val ... ... Glu CBMP-2B ... ... Ala ... ... Val ... ...Glu GDF-1 Asp Leu ... ... ... Val ... Ala Arg  65  70 hOP-1 Leu Asn AlaIle Ser Val Leu Tyr Phe mOP-1 ... ... ... ... ... ... ... ... ... hOP-2.. Ser ... Thr ... ... ... ... Tyr mOP-2 ... Ser ... Thr ... ... ... ...Tyr Vg1 Met Ser Pro ... ... Met ... Phe Tyr Vgr-1 Val ... ... ... ...... ... ... DPP ... Asp Ser Val Ala Met ... ... Leu CBMP-2A ... Ser ...... ... Met ... ... Leu CBMP-2B ... Ser ... ... ... Met ... ... LeuGDF-1 ... Ser Pro ... ... ... ... Phe ...  75  80 hOP-1 Asp Asp Ser SerAsn Val Ile Leu Lys mOP-1 ... ... ... ... ... ... ... ... ... hOP-2 ...Glu ... Asn ... ... ... ... Arg mOP-2 ... Ser ... Asn ... ... ... ...Arg DPP Asn ... Gln ... Thr ... Val ... ... Vg1 ... ... Asn Asp ... ...Val ... Arg Vgr-1 ... ... Asn ... ... ... ... ... ... CBMP-2A ... GluAsn Glu Lys ... Val ... ... CBMP-2B ... Glu Tyr Asp Lys ... Val ... ...GDF-1 ... Asn ... Asp ... ... Val ... Arg  85 hOP-1 Lys Tyr Arg Asn MetVal Val Arg mOP-1 ... ... ... ... ... ... ... ... hOP-2 ... Ala ... ...... ... ... Lys mOP-2 ... His ... ... ... ... ... Lys DPP Asn ... GlnGlu ... Thr ... Val Vg1 His ... Glu ... ... Ala ... Asp Vgr-1 ... ...... ... ... ... ... ... CBMP-2A Asn ... Gln Asp ... ... ... Glu CBMP-2BAsn ... Gln Glu ... ... ... Glu GDF-1 Gln ... Glu Asp ... ... ... Asp 90  95 hOP-1 Ala Cys Gly Cys His mOP-1 ... ... ... ... ... hOP-2 ...... ... ... ... mOP-2 ... ... ... ... ... DPP Gly ... ... ... Arg Vg1Glu ... ... ... Arg Vgr-1 ... ... ... ... ... CBMP-2A Gly ... ... ...Arg CBMP-2B Gly ... ... ... Arg GDF-1 Glu ... ... ... Arg 100

[0050] Table III, set forth below, compares the amino acid sequence datafor six related biosynthetic constructs designated COPs 1, 3, 4, 5, 7,and 16. As with Table II, the dots mean that in that position there isan identical amino acid to that of COP-1, and dashes mean that the COP-1amino acid is missing at that position. TABLE III COP-1 Leu Tyr Val AspPhe Gln Arg Asp Val COP-3 ... ... ... ... ... ... ... ... ... COP-4 ...... ... ... ... Ser --- ... ... COP-5 ... ... ... ... ... Ser --- ...... COP-7 ... ... ... ... ... Ser --- ... ... COP-16 ... ... ... ... ...Ser --- ... ... 1  5 COP-1 Gly Trp Asp Asp Trp Ile Ile Ala COP-3 ... ...... ... ... ... Val ... COP-4 ... ... ... ... ... ... Val ... COP-5 ...... ... ... ... ... Val ... COP-7 ... ... Asn ... ... ... Val ... COP-16... ... Asn ... ... ... Val ... 10 15 COP-1 Pro Val Asp Phe Asp Ala TyrTyr COP-3 ... Pro Gly Tyr Gln ... Phe ... COP-4 ... Pro Gly Tyr Gln ...Phe ... COP-5 ... Pro Gly Tyr Gln ... Phe ... COP-7 ... Pro Gly Tyr His... Phe ... COP-16 ... Pro Gly Tyr Gln ... Phe ...  20  25 COP-1 Cys SerGly Ala Cys Gln Phe Pro COP-3 ... ... ... ... ... ... ... ... COP-4 ...... ... ... ... ... ... ... COP-5 ... His ... Glu ... Pro ... ... COP-7... His ... Glu ... Pro ... ... COP-16 ... His ... Glu ... Pro ... ... 30 COP-1 Ser Ala Asp His Phe Asn Ser Thr COP-3 ... ... ... ... ... ...... ... COP-4 ... ... ... ... ... ... ... ... COP-5 Leu ... ... ... ...... ... ... COP-1 Leu ... ... ... Leu ... ... ... COP-16 Leu ... ... ...... ... ... ... 35 40 COP-1 Asn His Ala Val Val Gln Thr Leu Val COP-3... ... ... ... ... ... ... ... ... COP-4 ... ... ... ... ... ... ...... ... COP-5 ... ... ... ... ... ... ... ... ... COP-7 ... ... ... ...... ... ... ... ... COP-16 ... ... ... ... ... ... ... ... ... 45 50COP-1 Asn Asn Met Asn Pro Gly Lys Val COP-3 ... ... ... ... ... ... ...... COP-4 ... ... ... ... ... ... ... ... COP-5 ... Ser Val ... Ser LysIle ... COP-1 ... Ser Val ... Ser Lys Ile ... COP-16 ... Ser Val ... SerLys Ile ... 55 COP-1 Pro Lys Pro Cys Cys Val Pro Thr COP-3 ... ... ...... ... ... ... ... COP-4 ... ... ... ... ... ... ... ... COP-5 ... ...Ala ... ... ... ... ... COP-1 ... ... Ala ... ... ... ... ... COP-16 ...... Ala ... ... ... ... ... 60 65 COP-1 Glu Leu Ser Ala Ile Ser Met LeuCOP-3 ... ... ... ... ... ... ... ... COP-4 ... ... ... ... ... ... ...... COP-5 ... ... ... ... ... ... ... ... COP-7 ... ... ... ... ... ...... ... COP-16 ... ... ... ... ... ... ... ... 70 COP-1 Tyr Leu Asp GlueAsn Ser Thr Val COP-3 ... ... ... ... ... Glu Lys ... COP-4 ... ... ...... ... Glu Lys ... COP-5 ... ... ... ... ... Glu Lys ... COP-7 ... ...... ... ... Glu Lys ... COP-16 ... ... ... ... ... Glu Lys ... 75 80COP-1 Val Leu Lys Asn Tyr Gln Glu Met COP-3 ... ... ... ... ... ... ...... COP-4 ... ... ... ... ... ... ... ... COP-5 ... ... ... ... ... ...... ... COP-7 ... ... ... ... ... ... ... ... COP-16 ... ... ... ... ...... ... ... 85 90 COP-1 Thr Val Val Gly Cys Gly Cys Arg COP-3 Val ...Glu ... ... ... ... ... COP-4 Val ... Glu ... ... ... ... ... COP-5 Val... Glu ... ... ... ... ... COP-7 Val ... Glu ... ... ... ... ... COP-16Val ... Glu ... ... ... ... ... 95

[0051] As is apparent from the foregoing amino acid sequencecomparisons, significant amino acid changes can be made within thegeneric sequences while retaining the morphogenic activity. For example,the GDF-1 protein shares approximately 70% amino acid sequence homologywith the collection of sequences defined by Table II.

[0052] It now has been discovered that the family of proteins describedby these sequences also is capable of initiating and maintaining thetissue-specific developmental cascade in tissues other than bone andbone cartilage. When combined with naive progenitor cells as disclosedherein, these proteins, termed morphogens, are capable of inducing theproliferation and differentiation of the progenitor cells. In thepresence of appropriate tissue-specific signals to direct thedifferentiation of these cells, and a morphogenically permissiveenvironment, these morphogens are capable of reproducing the cascade ofcellular and molecular events that occur during embryogenesisdevelopment to yield adult, functioning tissue.

[0053] A key to these developments was the creation of a mammaliantissue model system, namely a model system for endochondral boneformation, and investigation of the cascade of events important for bonetissue morphogenesis. Work on this system has enabled discovery not onlyof bone inductive morphogens, but also of tissue inductive morphogensand their activities. The methods used to develop the bone model system,now well known in the art, along with the proteins of this invention,can be used to create model systems for other tissues, such as liver(see infra).

[0054] Using the model system for endochondral bone formation, it alsohas been discovered that the local environment in which the morphogenicmaterial is placed is important for tissue morphogenesis. As usedherein, “local environment” is understood to include the tissuestructural matrix and the environment surrounding the tissue. Forexample, in addition to needing an appropriate anchoring substratum fortheir proliferation, the morphogen-stimulated cells need signals todirect the tissue-specificity of their differentiation. These signalsvary for the different tissues and may include cell surface markers. Inaddition, vascularization of new tissue requires a local environmentwhich supports vascularization. Using the bone model system as anexample, it is known that, under standard assay conditions, implantingosteoinductive morphogens into loose mesenchyme in the absence of atissue-specifying matrix generally does not result in endochondral boneformation unless very high concentrations of the protein are implanted.By contrast, implanting relatively low concentrations of the morphogenin association with a suitably modified bone-derived matrix is resultsin the formation of fully functional endochondral bone (see, forexample, Sampath et al. (1981) PNAS 78:7599-7603 and U.S. Pat. No.4,975,526). In addition, a synthetic matrix comprised of a structuralpolymer such as tissue-specific collagen and tissue-specific cellattachment factors such as tissue-specific glycosylaminoglycans, willallow endochondral bone formation (see, for example, U.S. Ser. No.529,582, filed May 30, 1990, incorporated herein by reference). Finally,if the morphogen and a suitable bone or bone cartilage-specific matrix(e.g., comprising Type I cartilage) are implanted together in loosemesenchyme, bone cartilage and endochondral bone formation will result,including the formation of bone marrow and a vascular system. However,if the same composition is provided to a nonvascular environment, suchas to cultured cells in vitro or at an cartilage-specific locus, tissuedevelopment does not continue beyond cartilage formation (see infra).Similarly, a morphogenic composition containing a cartilage-specificmatrix composed of Type 2 collagen is expected to induce formation ofnon-bone cartilage tissue in vivo (e.g., hyaline). However, if thecomposition is provided to a vascular-supporting environment, such asloose mesenchyme, the composition is capable of inducing thedifferentiation of proliferating progenitor cells into chondrocytes andosteoblasts, resulting in bone formation.

[0055] It also has been discovered that tissue morphogenesis requires amorphogenically permissive environment. Clearly, in fully-functioninghealthy tissue that is not composed of a permanently renewing cellpopulation, there must exist signals to prevent continued tissue growth.Thus, it is postulated that there exists a control mechanism, such as afeedback control mechanism, which regulates the control of cell growthand differentiation. In fact, it is known that both TGF-β, and MIS arecapable of inhibiting cell growth when present at appropriateconcentrations. In addition, using the bone model system it can be shownthat osteogenic devices comprising a bone-derived carrier (matrix) thathas been demineralized and guanidine-extracted to substantially removethe noncollagenous proteins does allow endochondral bone formation whenimplanted in association with an osteoinductive morphogen. If, however,the bone-derived carrier is not demineralized but rather is washed onlyin low salt, for example, induction of endochondral bone formation isinhibited, suggesting the presence of one or more inhibiting factorswithin the carrier.

[0056] Another key to these developments was determination of the broaddistribution of these morphogens in developing and adult tissue. Forexample, DPP is expressed in both embryonic and developing Drosophilatissue. Vgl has been identified in Xenopus embryonic tissue. Vgr-1transcripts have been identified in a variety of murine tissues,including embryonic and developing brain, lung, liver, kidney andcalvaria (dermal bone) tissue. Recently, Vgr-1 transcripts also havebeen identified in adult murine lung, kidney, heart, and brain tissue,with especially high abundance in the lung (see infra).

[0057] OP-1 and the CBMP2 proteins, both first identified as bonemorphogens, have been identified in mouse and human placenta,hippocampus, calvaria and osteosarcoma tissue as determined byidentification of OP-1 and CMBP2-specific sequences in cDNA librariesconstructed from these tissues (see U.S. Ser. No. 422,699, incorporatedherein by reference). Additionally, the OP-1 protein is present in avariety of embryonic and developing tissues including kidney, liver,heart, adrenal tissue and brain as determined by Western blot analysisand immunolocalization (see infra and Attorney Docket No. CRP-058).OP-1-specific transcripts also have been identified in both embryonicand developing tissues, most abundantly in developing kidney, bladderand brain (see infra). OP-1 also has been identified as a mesoderminducing factor present during embryogenesis (see infra). Moreover, OP-1has been shown to be associated with in satellite muscle cells andassociated with pluripotential stem cells in bone marrow followingdamage to adult murine endochondral bone, indicating its morphogenicrole in tissue repair and regeneration. In addition, a novel proteinGDF-1 (see Table II) has been identified in neural tissue (Lee, (1991)PNAS 88 4250-4254).

[0058] Exemplification

Identification and Isolation of Morphogens

[0059] Among the proteins useful in this invention are proteinsoriginally identified as bone inductive proteins, such as the OP-1, OP-2and the CBMP proteins, as well as amino acid sequence related proteinssuch as DPP (from Drosophila), Vgl (from Xenopus) and Vgr-1 (from mouse,see Table II and Sequence Listing). The members of this family, whichinclude some members of the TGF-β super family of structurally relatedproteins, share substantial amino acid sequence homology in theirC-terminal regions. The OP-2 proteins have an extra cysteine residue inthis region (position 41), in addition to the conserved cysteineskeleton in common with the other proteins in this family. The proteinsare inactive when reduced, but are active as oxidized homodimers and asvarious oxidized heterodimers.

[0060] Accordingly, the morphogens of this invention can be described byeither of the following two species of generic amino acid sequences:Generic Sequence 1 or Generic Sequence 2, (Seq. ID Nos. 1 and 2), whereeach Xaa indicates one of the 20 naturally-occurring L-isomer, α-aminoacids or a derivative thereof. Particularly useful sequences that fallwithin this family of proteins include the 102 C-terminal residues ofVgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, and CBMP-2B, as well as theirintact mature amino acid sequences. 7-19). In addition, biosyntheticconstructs designed from the generic sequences, such as COP-1, COP-3-5,COP-7, and COP-16 also are useful.

[0061] Generic sequences showing preferred amino acids compiled fromsequences identified to date as useful as morphogens (e.g., Tables IIand III) are described as: Generic Sequence 3 (Seq. ID No. 3) andGeneric Sequence 4 (Seq. ID No. 4). Note that these generic sequenceshave a 7 or 8-cysteine skeleton where inter- or intramolecular disulfidebonds can form, and contain certain critical amino acids which influencethe tertiary structure of the proteins. It is also possible that thediffering N-termini of the naturally occurring proteins provide atissue-specific or other, important modulating activity of theseproteins.

[0062] Given the foregoing amino acid and DNA sequence information, thelevel of skill in the art, and the disclosure of U.S. Pat. No. 5,011,691and published PCT specification US 89/01469, published Oct. 19, 1989,the disclosures of which are incorporated herein by reference, variousDNAs can be constructed which encode at least the minimally requiredactive domain of a morphogen of this invention, and various analogsthereof (including allelic variants and those containing geneticallyengineered mutations), as well as fusion proteins, truncated forms ofthe mature proteins, deletion and insertion mutants, and similarconstructs. Moreover, DNA hybridization probes can be constructed fromfragments of the genes encoding any of these proteins, or designed denovo from the generic sequence. These probes then can be used to screendifferent genomic and cDNA libraries to identify additional morphogenicproteins from different tissues.

[0063] The DNAs can be produced by those skilled in the art using wellknown DNA manipulation techniques involving genomic and cDNA isolation,construction of synthetic DNA from synthesized oligonucleotides, andcassette mutagenesis techniques. 15-100 mer oligonucleotides may besynthesized on a Biosearch DNA Model 8600 Synthesizer, and purified bypolyacrylamide gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer.The DNA then may be electroeluted from the gel. Overlapping oligomersmay be phosphorylated by T4 polynucleotide kinase and ligated intolarger blocks which also may be purified by PAGE.

[0064] The DNA from appropriately identified clones then can beisolated, subcloned (preferably into an expression vector), andsequenced. Plasmids containing sequences of interest then can betransfected into an appropriate host cell for expression of themorphogen and further characterization. The host may be a procaryotic oreucaryotic cell since the former's inability to glycosylate protein willnot destroy the protein's morphogenic activity. Useful host cellsinclude E. coli, Saccharomyces, the insect/baculovirus cell system,myeloma cells, and various other mammalian cells. The vectorsadditionally may encode various sequences to promote correct expressionof the recombinant protein, including transcription promoter andtermination sequences, enhancer sequences, preferred ribosome bindingsite sequences, preferred mRNA leader sequences, preferred signalsequences for protein secretion, and the like.

[0065] The DNA sequence encoding the gene of interest also may bemanipulated to remove potentially inhibiting sequences or to minimizeunwanted secondary and tertiary structure formation. The recombinantmorphogen also may be expressed as a fusion protein. After beingtranslated, the protein may be purified from the cells themselves orrecovered from the culture medium. All biologically active protein formscomprise dimeric species joined by disulfide bonds or otherwiseassociated, produced by refolding and oxidizing one or more of thevarious recombinant polypeptide chains within an appropriate eucaryoticcell or in vitro after expression of individual subunits. A detaileddescription of morphogens expressed from recombinant DNA in E. coli isdisclosed in U.S. Ser. No. 422,699 filed Oct. 17, 1989, the disclosureof which is incorporated herein by reference. A detailed description ofmorphogens expressed from recombinant DNA in numerous differentmammalian cells is disclosed in U.S. Ser. No. 569,920 filed Aug. 20,1990, the disclosure of which is hereby incorporated by reference.

[0066] Alternatively, morphogenic polypeptide chains can be synthesizedchemically using conventional peptide synthesis techniques well known tothose having ordinary skill in the art. For example, the proteins may besynthesized intact or in parts on a Biosearch solid phase peptidesynthesizer, using standard operating procedures. Completed chains thenare deprotected and purified by HPLC (high pressure liquidchromatography). If the protein is synthesized in parts, the parts maybe peptide bonded using standard methodologies to form the intactprotein. In general, the manner in which the morphogens are made can beconventional and does not form a part of this invention.

Morphogen Distribution

[0067] The generic function of the morphogens of this inventionthroughout the life of the organism can be evidenced by their expressionin a variety of disparate mammalian tissues. Determination of the tissuedistribution of morphogens also may be used to identify differentmorphogens expressed in a given tissue, as well as to identify new,related morphogens. The proteins (or their mRNA transcripts) are readilyidentified in different tissues using standard methodologies and minormodifications thereof in tissues where expression may be low. Forexample, protein distribution may be determined using standard Westernblot analysis or immunofluorescent techniques, and antibodies specificto the morphogen or morphogens of interest. Similarly, the distributionof morphogen transcripts may be determined using standard Northernhybridization protocols and transcript-specific probes.

[0068] Any probe capable of hybridizing specifically to a transcript,and distinguishing the transcript of interest from other, relatedtranscripts may be used. Because the morphogens of this invention sharesuch high sequence homology in their active, C-terminal domains, thetissue distribution of a specific morphogen transcript may best bedetermined using a probe specific for the pro region of the immatureprotein and/or the N-terminal region of the mature protein. Anotheruseful sequence is the 3′ non-coding region flanking and immediatelyfollowing the stop codon. These portions of the sequence varysubstantially among the morphogens of this invention, and accordingly,are specific for each protein. For example, a particularly usefulVgr-1-specific probe sequence is the PvuII-SacI fragment, a 265 bpfragment encoding both a portion of the untranslated pro region and theN-terminus of the mature sequence (see Lyons et al. (1989) PNAS86:4554-4558 for a description of the cDNA sequence). Similarly,particularly useful mOP-1-specific probe sequences are the BstX1-BglIfragment, a 0.68 Kb sequence that covers approximately two-thirds of themOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb sequence immediatelyupstream of the 7-cysteine domain; and the Ear1-Pst1 fragment, an 0.3 Kbfragment containing a portion of the 3′untranslated sequence (See Seq.ID No. 15).

[0069] Using these morphogen-specific probes, which may be syntheticallyengineered or obtained from cloned sequences, morphogen transcripts canbe identified in mammalian tissue, using standard methodologies wellknown to those having ordinary skill in the art. Briefly, total RNA isprepared from various adult murine tissues (e.g., liver, kidney, testis,heart, brain, thymus and stomach) by a stand and methodology such as bythe method of Chomczyaski et al. ((1987) Anal. Biochem 162:156-159) anddescribed below. Poly (A)+ RNA is prepared by using oligo (dT)-cellulosechromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).Poly (A)+ RNA (generally 15 μg) from each tissue is fractionated on a 1%agarose/formaldehyde gel and transferred onto a Nytran membrane(Schleicher & Schuell). Following the transfer, the membrane is baked at80° C. and the RNA is cross-linked under UV light (generally 30 secondsat 1 mW/cm²). Prior to hybridization, the appropriate probe (e.g., thePvuII-SacI Vgr-1 fragment) is denatured by heating. The hybridization iscarried out in a lucite cylinder rotating in a roller bottle apparatusat approximately 1 rev/min for approximately 15 hours at 37° C. using ahybridization mix of 40% formamide, 5× Denhardts, 5×SSPE, and 0.1% SDS.Following hybridization, the non-specific counts are washed off thefilters in 0.1×SSPE, 0.1% SDS at 50° C. Northern blots performed usingVgr-1 probes specific to the variable N terminus of the mature sequenceindicate that the Vgr-1 message is approximately 3.5 Kb.

[0070]FIG. 1 is a photomicrograph representing a Northern blot analysisprobing a number of adult murine tissues with the Vgr-1 specific probes:liver, kidney, testis, heart, brain, thymus and stomach, represented inlanes 3-10, respectively. Lanes 1 and 12 are size standards and lanes 2and 11 are blank. Among the tissues tested, Vgr-1 appears to beexpressed most abundantly in adult lung, and to a lesser extent in adultkidney, heart and brain. These results confirm and expand on earlierstudies identifying Vgr-1 and Vgr-1-like transcripts in severalembryonic and adult murine tissue (Lyons et al. (1989) PNAS86:4554-4558), as well as studies identifying OP-1 and CBMP2 in varioushuman cDNA libraries (e.g., placenta, hippocampus, calvaria, andosteosarcoma, see U.S. Ser. No. 422,699, filed Oct. 17, 1989, andOzkaynak et al., (1990) EMBO 9:2085-2093).

[0071] Using the same general probing methodology, mOP-1 transcriptsalso have been identified in a variety of murine tissues, includingembryo and various developing tissues, as can be seen in FIGS. 2 and 3.Details of the probing methodology are disclosed in copending (AttorneyDocket CRP058), the disclosure of which is incorporated herein. TheNorthern blots represented in FIG. 2 probed RNA prepared from developingbrain, spleen, lung, kidney (and adrenal gland), heart, and liver in 13day post natal mice (panel A) or 5 week old mice (panel B). The OP-1specific probe was a probe containing the 3′ untranslated sequencesdescribed supra (0.34 Kb EarI-Pst I fragment). As a control for RNArecovery, EF-Tu (translational elongation factor) mRNA expression alsowas measured (EF-Tu expression is assumed to be relatively uniform inmost tissues).

[0072] The arrowheads indicate the OP-1 specific messages observed inthe various tissues. As can be seen in FIG. 2, OP-1 expression levelsvary significantly in the spleen, lung, kidney and adrenal tissues,while the EF-Tu mRNA levels are constant. Uniformly lower levels ofEF-Tu mRNA levels were found in the heart, brain and liver. As can beseen from the photomicrograph, the highest levels of OP-1 mRNA appear tobe in kidney and adrenal tissue, followed by the brain. By contrast,heart and liver did not give a detectable signal. Not shown areadditional analyses performed on bladder tissue, which shows significantOP-1 mRNA expression, at levels close to those in kidney/adrenal tissue.The Northern blots also indicate that, like GDF-1, OP-1 mRNA expressionmay be bicistonic in different tissues. Four transcripts can be seen: 4Kb, 2.4 Kb, 2.2 Kb, and 1.8 Kb transcripts can be identified in thedifferent tissues, and cross probing with OP-1 specific probes from theproregion and N-terminal sequences of the gene indicate that thesetranscripts are OP-1 specific.

[0073] A side by side comparison of OP-1 and Vgr-1 in FIG. 3 shows thatthe probes distinguish between the morphogens Vgr-1 and OP-1 transcriptsin the different tissues, and also highlights the multiple transcriptionof OP-1 in different tissues. Specifically, FIG. 3 compares theexpression of OP-1 (Panels B and D), Vgr-1 (Panel C) and EF-Tu (Panel A)(control) mRNA in 17 day embryos (lane 1) and 3 day post-natal mice(lane 2). The same filter was used for sequential hybridizations withlabeled DNA probes specific for OP-1 (Panels B and D), Vgr-i (Panel C),and EF-Tu (Panel A). Panel A: the EF-Tu specific probe (control) was the0.4 Kb HindIII-SacI fragment (part of the protein coding region), theSacI site used belonged to the vector; Panel B: the OP-1 specific probewas the 0.68 Kb BstXI-BglI fragment containing pro region sequences;Panel D; the OP-1 specific probe was the 0.34 Kb EarI-PstI fragmentcontaining the 3′ untranslated sequence; Panel C: the Vgr-1 specificprobe was the 0.26 Kb PvuII-SacI fragment used in the Vgr-1 blotsdescribed above.

[0074] The 1.8-2.5 Kb OP-1 mRNA appears approximately two times higherin three day post natal mice than in 17 day embryos, perhaps reflectingphases in bone and/or kidney development. In addition, of the fourmessages found in brain, the 2.2 Kb transcript appears most abundant,whereas in lung and spleen the 1.8 Kb message predominates. Finally,careful separation of the renal and adrenal tissue in five week old micereveals that the 2.2 Kb transcripts were derived from renal tissue andthe 4 Kb mRNA is more prominent in adrenal tissue (see FIG. 2).

[0075] Similarly, using the same general probing methodology, BMP3 andCBMP2B transcripts recently have been identified in abundance in lungtissue.

[0076] Morphogen distribution in embryonic tissue can be determinedusing five or six-day old mouse embryos and standard immunofluorescencetechniques in concert with morphogen-specific antisera. For example,rabbit anti-OP-1 antisera is readily obtained using any of a number ofstandard antibody protocols well known to those having ordinary skill inthe art. The antibodies then are fluorescently labelled using standardprocedures. A five or six-day old mouse embryo then is thin-sectionedand the various developing tissues probed with the labelled antibody,again following standard protocols. Using this technique, OP-1 proteinis detected in developing brain and heart.

[0077] This method also may be used to identify morphogens in adulttissues undergoing repair. For example, a fracture site can be inducedin a rat long bone such as the femur. The fracture then is allowed toheal for 2 or 3 days. The animal then is sacrificed and the fracturedsite sectioned and probed for the presence of the morphogen e.g., OP-1,with fluorescently labelled rabbit anti-OP-1 antisera using standardimmunblocalization methodology. This technique identifies OP-1 in musclesatellite cells, the progenitor cells for the development of muscle,bone cartilage and endochondral bone. In addition, OP-1 is detected withpotential pluripotential stem cells in the bone marrow, indicating itsmorphogenic role in tissue repair and regeneration.

[0078] OP-1 protein also has been identified in rat brain using standardimmunofluorescence staining technique. Specifically, adult rat brain(2-3 months old) and spinal cord is frozen and sectioned. Anti-OP-1,raised in rabbits and purified on an OP-1 affinity column prepared usingstandard methodologies, was added to the sections under standardconditions for specific binding. Goat anti-rabbit IgG, labelled withfluorescence, then was used to visualize OP-1 antibody binding to tissuesections.

[0079] As can be seen in FIGS. 4A and 4B, immunofluorescence stainingdemonstrates the presence of OP-1 in adult rat CNS. Similar andextensive staining is seen in both the brain (4A) and spinal cord (4B).OP-1 appears to be predominantly localized to the extracellular matrixof the grey matter, present in all areas except the neuronal cellbodies. In white matter, staining appears to be confined to astrocytes.A similar staining pattern also was seen in newborn rat (10 day old)brain sections.

Cell Differentiation

[0080] The ability of morphogens of this invention to induce celldifferentiation can be determined by culturing early mesenchymal cellsin the presence of the morphogen and then studying the histology of thecultured cells by staining with toluidine blue. For example, it is knownthat rat mesenchymal cells destined to become mandibular bone, whenseparated from the overlying epithelial cells at stage 11 and culturedin vitro under standard tissue culture conditions, will not continue todifferentiate. However, if these same cells are left in contact with theoverlying endoderm for an additional day, at which time they becomestage 12 cells, they will continue to differentiate on their own invitro to form chondrocytes. Further differentiation into obsteoblastsand, ultimately, mandibular bone, requires an appropriate localenvironment, e.g., a vascularized environment.

[0081] It has now been discovered that stage 11 mesenchymal cells,cultured in vitro in the presence of a morphogen, e.g., OP-1, continueto differentiate in vitro to form chondrocytes. These stage 11 cellsalso continue to differentiate in vitro if they are cultured with thecell products harvested from the overlying endodermal cells. Moreover,OP-1 can be identified in the medium conditioned by endodermal cellseither by Western blot or immunofluorescence. This experiment may beperformed with other morphogens and with different mesenchymal cells toassess the cell differentiation capability of different morphogens, aswell as their distribution in different developing tissues.

[0082] As another example of morphogen-induced cell differentiation, theeffect of OP-1 on the differentiation of neuronal cells has been testedin culture. Specifically, the effect of OP-1 on the NG108-5neuroblastoma x glioma hybrid clonal cell line has been assessed. Thecell line shows a fibroblastic-type morphology in culture. The cell linecan be induced to differentiate chemically using 0.5 mM butyrate, 1%DMSO or 500 mM Forskolin, inducing the expression of virtually allimportant neuronal properties of cultured primary neurons. However,chemical induction of these cells also induces cessation of celldivision.

[0083] In the present experiment NG108-5 cells were subcultured onpoly-L-lysine coated 6 well plates. Each well contained 40-50,000 cellsin 2.5 ml of chemically defined medium. On the third day 2.5 μl of OP-1in 60% ethanol containing 0.025% trifluoroacetic was added to each well.OP-1 concentrations of 0, 1, 10, 40 and 100 ng/ml were tested. The mediawas changed daily with new aliquots of OP-1. After four days with 40 and100 ng OP-1/ml concentrations, OP-1 induced differentiation of NG108cells. FIG. 5 shows the morphological changes that occur. The OP-1induces clumping and rounding of the cells and the production of neuriteoutgrowths (processes). Compare FIG. 5A (naive NG108 cells) with FIG.5B, showing the effects of OPI-treated cells. Thus the OP-1 can inducethe cells to differentiate into a neuronal cell morphology. Some of theoutgrowths appear to join in a synaptic-type junction. This effect wasnot seen in cells incubated with TGF-B1 at concentrations of 1 to 100ng/ml.

[0084] The neuroprotective effects of OP-1 were demonstrated bycomparison with chemical differentiation agents on the NG108 cells.50,000 cells were plated on 6 well plates and treated with butyrate,DMSO, Forskolin or OP-1 for four days. Cell counts demonstrated that inthe cultures containing the chemical agents the differentiation wasaccompanied by a cessation of cell division. In contrast, the cellsinduced to differentiate by OP-1 continued to divide, as determined byH³-thymidine uptake. The data suggest that OP-1 is capable ofmaintaining the stability of the cells in culture after differentiation.

[0085] As yet another, related example, the ability of the morphogens ofthis invention to induce the “redifferentiation” of transformed cellsalso has been assessed. Specifically, the effect of OP-1 on human ECcells (embryo carcinoma cells, NTERA-Z CL.D1) is disclosed herein. Inthe absence of an external stimulant these cells can be maintained asundifferentiated stem cells, and can be induced to grow in serum freemedia (SFM). In the absence of morphogen treatment the cells proliferaterampantly and are anchorage-independent. The effect of morphogentreatment is seen in FIGS. 6A-D. FIGS. 6A and 6B show 4 days of growthin SFM in the presence of OP-1 (25 ng/ml, 6A) or the absence ofmorphogen (6B). FIGS. 6C and 6D are 5 days growth in the presence of 10ng/ml OP-1 (6C) or no morphogen (6D). FIGS. 6C and 6D are at 10× and 20×magnification compared to FIGS. 6A and 5B. As can readily be seen, inthe presence of OP-1, EC cells grow as flattened cells, becominganchorage dependent. In addition, growth rate is reduced approximately10 fold. Finally, the cells are induced to differentiate.

Maintenance of Phenotype

[0086] The morphogens of this invention also may be used to maintain acell's differentiated phenotype. This morphogenic capability isparticularly useful for inducing the continued expression of phenotypein senescent or quiescent cells.

[0087] The phenotypic maintenance capability of morphogens is readilyassessed. A number of differentiated cells become senescent or quiescentafter multiple passages under standard tissue culture conditions invitro. However, if these cells are cultivated in vitro in associationwith a morphogen of this invention, the cells are induced to maintainexpression of their phenotype through multiple passages. For example,the alkaline phosphatase activity of cultured osteoblasts, like culturedosteoscarcoma cells and calvaria cells, is significantly reduced aftermultiple passages in vitro. However, if the cells are cultivated in thepresence of a morphogen (e.g., OP-1), alkaline phosphatase activity ismaintained over extended periods of time. Similarly, phenotypicexpression of myocytes also is maintained in the presence of themorphogen. This experiment may be performed with other morphogens anddifferent cells to assess the phenotypic maintenance capability ofdifferent morphogens on cells of differing origins.

[0088] Phenotypic maintenance capability also may be assessed in vivo,using a rat model for osteoporosis, disclosed in co-pending (Atty.Docket No. CRP-060), incorporated herein by reference. As disclosedtherein, Long Evans rats are ovariectomized to produce an osteoporoticcondition resulting from decreased estrogen production. Eight days afterovariectomy, rats are systemically provided with phosphate bufferedsaline (PBS) or OP-1 (21 μg or 20 μg) for 22 days. The rats then aresacrificed and and serum alkaline phosphatase levels, serum calciumlevels, and serum osteocalcin levels determined, using standardmethodologies. Three-fold higher levels of osteocalcin levels are foundin rats provided with 1 or 20 μg of OP-1. Increased alkaline phosphataselevels also were seen. Histomorphometric analysis on the tibialdiaphysical bone shows OP-1 can reduce bone mass lost due to the drop inestrogen levels.

Cell Stimulation

[0089] The ability of the morphogens of this invention to stimulate theproliferation of progenitor cells also can be assayed readily in vitro.Useful naive stem cells include pluripotential stem cells, which may beisolated from bone marrow or umbilical cord blood using conventionalmethodologies, (see, for example, Faradji et al., (1988) Vox Sanq. 55(3):133-138 or Broxmeyer et al., (1989) PNAS 86 (10):3828-3832), as wellas naive stem cells obtained from blood. Alternatively, embryonic cells(e.g., from a cultured mesodermal cell line) may be useful.

[0090] Another method for obtaining progenitor cells and for determiningthe ability of morphogens to stimulate cell proliferation is to captureprogenitor cells from an in vivo source. For example, a biocompatiblematrix material able to allow the influx of migratory progenitor cellsmay be implanted at an in vivo site long enough to allow the influx ofmigratory progenitor cells. For example, a bone-derived,guanidine-extracted matrix, formulated as disclosed for example inSampath et al. ((1983) PNAS 80:6591-6595), or U.S. Pat. No. 4,975,526,may be implanted into a rat at a subcutaneous site, essentiallyfollowing the method of Sampath et al. (ibid). After three days theimplant is removed, and the progenitor cells associated with the matrixdispersed and cultured.

[0091] Progenitor cells, however obtained, then are incubated in vitrowith a suspected morphogen under standard cell culture conditions wellknown to those having ordinary skill in the art. In the absence ofexternal stimuli, the progenitor cells do not, or minimally proliferateon their own in culture. However, if the cells are cultured in thepresence of a morphogen, such as OP-1, they are stimulated toproliferate. Cell growth can be determined visually orspectrophotometrically using standard methods well known in the art.

Proliferation of Progenitor Cell Populations

[0092] Progenitor cells may be stimulated to proliferate in vivo or exvivo. The cells may be stimulated in vivo by injecting or otherwiseproviding a sterile preparation containing the morphogen into theindividual. For example, the hemopoietic pluripotential stem cellpopulation of an individual may be stimulated to proliferate byinjecting or otherwise providing an appropriate concentration of themorphogen to the individual's bone marrow.

[0093] Progenitor cells may be stimulated ex vivo by contactingprogenitor cells of the population to be enhanced with a morphogen understerile conditions at a concentration and for a time sufficient tostimulate proliferation of the cells. In general, a period of from about10 minutes to about 24 hours should be sufficient. The stimulated cellsthen are provided to the individual as, for example, by injecting thecells to an appropriate in vivo locus. Suitable biocompatible progenitorcells may be obtained by any of the methods known in the art ordescribed herein.

Regeneration of Damaged or Diseased Tissue

[0094] The morphogens of this invention may be used to repair diseasedor damaged mammalian tissue. The tissue to be repaired is preferablyassessed, and excess necrotic or interfering scar tissue removed asneeded, by surgical, chemical, ablating or other methods known in themedical arts.

[0095] The morphogen then may be provided directly to the tissue locusas part of a sterile, biocompatible composition, either by surgicalimplantation or injection. Alternatively, a sterile, biocompatiblecomposition containing morphogen-stimulated progenitor cells may beprovided to the tissue locus. The existing tissue at the locus, whetherdiseased or damaged, provides the appropriate matrix to allow theproliferation and tissue-specific differentiation of progenitor cells.In addition, a damaged or diseased tissue locus, particularly one thathas been further assaulted by surgical means, provides a morphogenicallypermissive environment. For some tissues, it is envisioned that systemicprovision of the morphogen will be sufficient.

[0096] In some circumstances, particularly where tissue damage isextensive, the tissue may not be capable of providing a sufficientmatrix for cell influx and proliferation. In these instances, it may benecessary to provide the morphogen or morphogen-stimulated progenitorcells to the tissue locus in association with a suitable, biocompatibleformulated matrix, prepared by any of the means described below. Thematrix preferably is tissue-specific, in vivo biodegradable, andcomprises particles having dimensions within the range of 70-850 μm,most preferably 150-420 μm.

[0097] The morphogens of this invention also may be used to prevent orsubstantially inhibit scar tissue formation following an injury. If amorphogen is provided to a newly injured tissue locus, it can inducetissue morphogenesis at the locus, preventing the aggregation ofmigrating fibroblasts into non-differentiated connective tissue. Themorphogen preferably is provided as a sterile pharmaceutical preparationinjected into the tissue locus within five hours of the injury. Severalnon-limiting examples follow, illustrating the morphogens regeneratecapabilities in different issues. The proteins of this inventionpreviously have been shown to be capable of inducing cartilage andendochondral bone formation (See, for example U.S. Pat. No. 5,011,691).

[0098] As an example, protein-induced morphogenesis of substantiallyinjured liver tissue following a partial hepatectomy is disclosed.Variations on this general protocol may be used to test morphogenactivity in other different tissues. The general method involvesexcising an essentially nonregenerating portion of a tissue andproviding the morphogen, preferably as a soluble pharmaceuticalpreparation to the excised tissue locus, closing the wound and examiningthe site at a future date. Like bone, liver has a potential toregenerate upon injury during post-fetal life.

[0099] Morphogen, (e.g., purified recombinant human OP-1, mature form,was solubilized (1 mg/ml) in 50% ethanol (or compatible solvent)containing 0.1% trifluoroacetic acid (or compatible acid). Theinjectable OP-1 solution was prepared by diluting one volume ofOP-1/solvent-acid stock solution with 9 volumes of 0.2% rat serumalbumin in sterile PBS (phosphate-buffered saline).

[0100] Growing rats or aged rats were anesthetized by using ketamine.Two of the liver lobes (left and right) were cut out (approximately{fraction (1/3)} of the lobe) and the OP-1 was injected locally atmultiple sites along the cut ends. The amount of OP-1 injected was 100μg in 100 of PBS/RSA injection buffer. Placebo samples are injectionbuffer without OP-1. Five rats in each group were used. The wound wasclosed and the rats were allowed to eat normal food and drink tap water.

[0101] After 12 days, the rats were sacrificed and liver regenerationwas observed visually. The photomigraph in FIG. 7 illustratesdramatically the regenerative effects of OP-1 on liver regeneration. TheOP-1-injected group showed complete liver tissue regeneration and showedno sign of any cut in the liver (animal 2). By contrast, the controlgroup into which only PBS only was injected, although some amount ofregeneration was seen, lack of complete liver regeneration was evident(animal 1). The incision remains in this sample.

[0102] As another example, the ability of the morphogens of thisinvention to induce dentinogenesis also was assessed. To date, theunpredictable response of dental pulp tissue to injury is a basicclinical problem in dentistry. Cynomolgus monkeys were chosen as primatemodels as monkeys are presumed to be more indicative of human dentalbiology than models based on lower non-primate mammals.

[0103] Using standard dental surgical procedures, small areas (e.g., 2mm) of dental pulps were surgically exposed by removing the enamel anddentin immediately above the pulp (by drilling) of sample teeth,performing a partial amputation of the coronal pulp tissue, inducinghemostasis, application of the pulp treatment, and sealing and fillingthe cavity by standard procedures.

[0104] Pulp treatments used were: OP-1 dispersed in a carrier matrix;carrier matrix alone and no treatment. Twelve teeth per animal (four foreach treatment) were prepared, and two animals were used. At four weeks,teeth were extracted and processed histologically for analysis of dentinformation, and/or ground to analyze dentin mineralization. FIG. 8illustrates dramatically the effect of morphogen on osteodentinreparation. FIG. 8A is a photomicrograph of the control treatment (PBS)and shows little or no reparation. FIG. 8B is a photomicrograph oftreatment with carrier alone, showing minimal reparation. By contrast,treatment with morphogen (FIG. 8C) shows significant reparation. Theresults of FIG. 8 indicate that OP-1-CM (OP-1 plus carrier matrix)reliably induced formation of reparative or osteodentin bridges onsurgically exposed healthy dental pulps. By contrast, pulps treated withcarrier matrix alone, or not treated failed to form reparative dentin.

[0105] As another example, the morphogen-induced regenerative effects oncentral nervous system (CNS) repair may be assessed using a rat brainstab model. Briefly, male Long Evans rats are anesthesized and the headarea prepared for surgery. The calvariae is exposed using standardsurgical procedures and a hole drilled toward the center of each lobeusing a 0.035 K wire, just piercing the calvariae. 25 μl solutionscontaining either morphogen (OP-1, 25 μg) or PBS then is provided toeach of the holes by Hamilton syringe. Solutions are delivered to adepth approximately 3 mm below the surface, into the underlying cortex,corpus callosum and hippocampus. The skin then is sutured and the animalallowed to recover.

[0106] Three days post surgery, rats are sacrificed by decapitation andtheir brains processed for sectioning. Scar tissue formation isevaluated by immunofluoresence staining for glial fibrillary acidicprotein, a marker protein for glial scarring, to qualitatively determinethe degree of scar formation. Sections also are probed with anti-OP-1antibodies to determine the presence of OP-1.

Morphogen Activity Modulation

[0107] Antibodies to morphogens of this invention have been identifiedin healthy human sera. In addition, implanting devices comprisingmorphogen (e.g., OP-1) have been discovered to induce an increase inanti-morphogen antibodies (e.g., anti, anti-OP antibodies). It isanticipated that these antibodies comprise part of the body's regulationof morphogen activity in vivo. The presence of the antibodies, andfluctuations in their levels, which are readily monitored, can provide auseful method for monitoring tissue stasis and tissue viability (e.g.,identification of a pathological state). For example, standardradioimmunoassays or ELISA may be used to detect and quantify antibodiesin sera. These antibodies may be raised against isolated morphogensusing standard methodologies.

Matrix Preparation

[0108] The morphogens of this invention may be implanted surgically,dispersed in a biocompatible, preferably in vivo biodegradable matrixappropriately modified to provide a structure in which the morphogen maybe dispersed and which allows the influx, differentiation andproliferation of migrating progenitor cells. The matrix also shouldprovide signals capable of directing the tissue specificity of thedifferentiating cells, as well as a morphogenically permissiveenvironment, being essentially free of growth inhibiting signals.

[0109] In the absence of these features the matrix does not appear to besuitable as part of a morphogenic composition. Recent studies onosteogenic devices (morphogens dispersed within a formulated matrix)using matrices formulated from polylactic acid and/or polyglycolic acidbiopolymers, ceramics (a-tri-calcium-phosphate), or hydroxyapatite showthat these materials, by themselves, are unable to provide theappropriate environment for inducing de novo endochondral bone formationin rats by themselves. In addition, matrices formulated fromcommercially available highly purified, reconstituted collagens ornaturally-derived non-bone, species-specific collagen (e.g., from rattail tendon) also are unsuccessful in inducing bone when implanted inassociation with an osteogenic protein. These matrices apparently lackspecific structurally-related features which aid in directing the tissuespecificity of the morphogen-stimulated, differentiating progenitorcells.

[0110] The formulated matrix may be shaped as desired in anticipation ofsurgery or may be shaped by the physician or technician during surgery.Thus, the material may be used in topical, subcutaneous,intraperitoneal, or intramuscular implants to repair tissue or to induceits growth de novo. The matrix preferably is biodegradable in vivo,being slowly absorbed by the body and replaced by new tissue growth, inthe shape or very nearly in the shape of the implant.

[0111] Details of how to make and how to use the matrices useful in thisinvention are disclosed below.

Tissue-Derived Matrices

[0112] Suitable biocompatible, in vivo biodegradable acellular matricesmay be prepared from naturally-occurring tissue. The tissue is treatedwith suitable agents to substantially extract the cellular,nonstructural components of the tissue. The agents also should becapable of extracting any growth inhibiting components associated withthe tissue. The resulting material is a porous, acellular matrix,substantially depleted in nonstructurally-associated components.

[0113] The matrix also may be further treated with agents that modifythe matrix, increasing the number of pores and micropits on itssurfaces. Those skilled in the art will know how to determine whichagents are best suited to the extraction of nonstructural components fordifferent tissues. For example, soft tissues such as liver and lung maybe thin-sectioned and exposed to a nonpolar solvent such as, forexample, 100% ethanol, to destroy the cellular structure of the tissueand extract nonstructural components. The material then is dried andpulverized to yield nonadherent porous particles. Structural tissuessuch as cartilage and dentin where collagen is the primary component maybe demineralized and extracted with guanidine, essentially following themethod of Sampath et al. (1983) PNAS 80:6591-6595. For example,pulverized and demineralized dentin is extracted with five volumes of 4M guanidine-HCl, 50 mM Tris-HCl, pH 7.0 for 16 hours at 4° C. Thesuspension then is filtered. The insoluble material that remains iscollected and used to fabricate the matrix. The material is mostlycollagenous in manner. It is devoid of morphogenic activity. The matrixparticles may further be treated with a collagen fibril-modifying agentthat extracts potentially unwanted components from the matrix, andalters the surface structure of the matrix material. Useful agentsinclude acids, organic solvents or heated aqueous media. A detaileddescription of these matrix treatments are disclosed in U.S. Pat. No.4,975,526 and copending U.S. Ser. No. 483,913, filed Feb. 22, 1990 andincorporated herein by reference.

[0114] After contact with the fibril-modifying agent, the treated matrixmay be washed to remove any extracted components, following a form ofthe procedure set forth below:

[0115] 1. Suspend matrix preparation in TBS (Tris-buffered saline) 1g/200 ml and stir at 4° C. for 2 hrs; or in 6 M urea, 50 mm Tris-HCl,500 mM NaCl, pH 7.0 (UTBS) or water and stir at room temperature (RT)for 30 minutes (sufficient time to neutralize the pH);

[0116] 2. Centrifuge and repeat wash step; and

[0117] 3. Centrifuge; discard supernatant; water wash residue; and thenlyophilize.

Synthetic Tissue-Specific Matrices

[0118] In addition to the naturally-derived tissue-specific matricesdescribed above, useful tissue-specific matrices may be formulatedsynthetically if appropriately modified. These porous biocompatible, invivo biodegradable synthetic matrices are disclosed in copending U.S.Ser. No. 529,852, filed May 30, 1990, the disclosure of which is herebyincorporated by reference. Briefly, the matrix comprises a porouscrosslinked structural polymer of biocompatible, biodegradable collagenand appropriate, tissue-specific glycosaminoglycans as tissue-specificcell attachment factors. Collagen derived from a number of sources maybe suitable for use in these synthetic matrices, including insolublecollagen, acid-soluble collagen, collagen soluble in neutral or basicaqueous solutions, as well as those collagens which are commerciallyavailable.

[0119] Glycosaminoglycans (GAGs) or mucopolysaccharides arehexosamine-containing polysaccharides of animal origin that have atissue specific distribution, and therefore may be used to helpdetermine the tissue specificity of the morphogen-stimulateddifferentiating cells. Reaction with the GAGs also provides collagenwith another valuable property, i.e., inability to provoke an immunereaction (foreign body reaction) from an animal host.

[0120] Chemically, GAGs are made up of residues of hexoaminesglycosidically bound and alternating in a more-or-less regular mannerwith either hexouronic acid or hexose moieties (see, e.g., Dodgson etal. in Carbohydrate Metabolism and its Disorders (Dickens et al., eds.)Vol. 1, Academic Press (1968)). Useful GAGs include hyaluronic acid,heparin, heparin sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate,dermatan sulfate, and keratin sulfate. Other GAGs are suitable forforming the matrix described herein, and those skilled in the art willeither know or be able to ascertain other suitable GAGs using no morethan routine experimentation. For a more detailed description ofmucopolysaccharides, see Aspinall, Polysaccharides, Pergamon Press,Oxford (1970). For example, as disclosed in U.S. application Se. No.529,852, chondroitin-6-sulfate can be used where endochondral boneformation is desired. Heparin sulfate, on the other hand, may be used toformulate synthetic matrices for use in lung tissue repair.

[0121] Collagen can be reacted with a GAG in aqueous acidic solutions,preferably in diluted acetic acid solutions. By adding the GAG dropwiseinto the aqueous collagen dispersion, coprecipitates of tangled collagenfibrils coated with GAG results. This tangled mass of fibers then can behomogenized to form a homogeneous dispersion of fine fibers and thenfiltered and dried.

[0122] Insolubility of the collagen-GAG products can be raised to thedesired degree by covalently crosslinking these materials, which alsoserves to raise the resistance to resorption of these materials. Ingeneral, any covalent cross-linking method suitable for cross-linkingcollagen also is suitable for crosslinking these composite materials,although crosslinking by a dehydrothermal process is preferred.

[0123] When dry, the crosslinked particles are essentially spherical,with diameters of about 500 μm. Scanning electron miscroscopy showspores of about 20 μm on the surface and 40 μm on the interior. Theinterior is made up of both fibrous and sheet-like structures, providingsurfaces for cell attachment. The voids interconnect, providing accessto the cells throughout the interior of the particle. The materialappears to be roughly 99.5% void volume, making the material veryefficient in terms of the potential cell mass that can be grown per gramof microcarrier.

[0124] The morphogens described herein can be combined and dispersed inan appropriately modified tissue-specific matrix using any of themethods described below:

[0125] 1. Ethanol Precipitation

[0126] Matrix is added to the morphogen dissolved in guanidine-HCl.Samples are vortexed and incubated at a low temperature. Samples arethen further vortexed. Cold absolute ethanol is added to the mixturewhich is then stirred and incubated. After centrifugation (microfuge,high speed) the supernatant is discarded. The matrix is washed with coldconcentrated ethanol in water and then lyophilized.

[0127] 2. Acetonitrile Trifluoroacetic Acid Lyophilization

[0128] In this procedure, morphogen in an acetonitrile trifluroaceticacid (ACN/TFA solution is added to the carrier material. Samples arevigorously vortexed many times and then lyophilized.

[0129] 3. Buffered Saline Lyophilization

[0130] Morphogen preparations in physiological saline may also bevortexed with the matrix and lyophilized to produce morphogenicallyactive material.

Bioassay

[0131] The following sets forth various procedures for evaluating the invivo morphogenic utility of the morphogens and morphogenic compositionsof this invention. The proteins and compositions may be injected orsurgically implanted in a mammal, following any of a number ofprocedures well known in the art. For example, surgical implantbioassays may be performed essentially following the procedure ofSampath et al. (1983) PNAS 80:6591-6595.

[0132] Histological Evaluation

[0133] Histological sectioning and staining is preferred to determinethe extent of morphogenesis in vivo, particularly in tissue repairprocedures. Excised implants are fixed in Bouins Solution, embedded inparaffin, and cut into 6-8 μm sections. Staining with toluidine blue orhemotoxylin/eosin demonstrates clearly the ultimate development of thenew tissue. Twelve day implants are usually sufficient to determinewhether the implants contain newly induced tissue.

[0134] Successful implants exhibit a controlled progression through thestages of induced tissue development allowing one to identify and followthe tissue-specific events that occur. For example, in endochondral boneformation the stages include: (1) leukocytes on day one; (2) mesenchymalcell migration and proliferation on days two and three; (3) chondrocyteappearance on days five and six; (4) cartilage matrix formation on dayseven; (5) cartilage calcification on day eight; (6) vascular invasion,appearance of osteoblasts, and formation of new bone on days nine andten; (7) appearance of osteoblastic and bone remodeling and dissolutionof the implanted matrix on days twelve to eighteen; and (8)hematopoietic bone marrow differentiation in the ossicle on daytwenty-one.

[0135] Biological Markers

[0136] In addition to histological evaluation, biological markers may beused as a marker for tissue morphogenesis. Useful markers includetissue-specific enzymes whose activities may be assayed (e.g.,spectrophotometrically) after homogenization of the implant. Theseassays may be useful for quantitation and for obtaining an estimate oftissue formation quickly after the implants are removed from the animal.For example, alkaline phosphatase activity may be used as a marker forosteogenesis.

[0137] Incorporation of systemically provided morphogens may be followedusing tagged morphogens (e.g., radioactively labelled) and determiningtheir localization in new tissue, and/or by monitoring theirdisappearance from the circulatory system using a standard pulse-chaselabeling protocol. The morphogen also may be provided with atissue-specific molecular tag, whose uptake may be monitored andcorrelated with the concentration of morphogen provided. As an example,ovary removal in female rats results in reduced bone alkalinephosphatase activity, rendering the rats predisposed to osteoporosis. Ifthe female rats now are provided with a morphogen, e.g., OP-1, areduction in the systemic concentration of calcium (CA²⁺) is seen, whichcorrelates with the presence of the provided morphogen and can be shownto correspond to increased alkaline phosphatase activity.

[0138] The invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Thepresent embodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

1 16 1 97 PRT Artificial Sequence VARIANT (1)..(97) wherein each Xaaindependently indicates one of the 20 natural L-isomers amino acids or aderivative thereof 1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa XaaCys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Cys Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Cys Xaa Cys 85 90 95 Xaa 2 97 PRT Artificial Sequence VARIANT(1)..(97) wherein each Xaa independently indicates one of the 20 naturalL-isomers amino acids or a derivative thereof 2 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Cys XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 85 90 95 Xaa 3 97 PRTArtificial Sequence VARIANT (1)..(97) wherein each Xaa is independentlyselected from a group of one or more specified amino acids as defined inthe specification 3 Leu Tyr Val Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa TrpXaa Xaa Ala 1 5 10 15 Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly XaaCys Xaa Xaa Pro 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala XaaXaa Xaa Xaa Leu 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa XaaXaa Xaa Xaa Xaa 65 70 75 80 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa ValXaa Xaa Cys Gly Cys 85 90 95 Xaa 4 102 PRT Artificial Sequence VARIANT(1)..(102) wherein each Xaa is independently selected from a group ofone or more specified amino acids as defined in the specification 4 CysXaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 1 5 10 15Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 30Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 7580 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 85 9095 Xaa Xaa Cys Gly Cys Xaa 100 5 139 PRT Homo sapiens tissue typehippocampus hOP1-MATURE 5 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn ArgSer Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn ValAla Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His GluLeu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile AlaPro Glu Gly Tyr Ala Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe ProLeu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln ThrLeu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys AlaPro Thr Gln Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp SerSer Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg AlaCys Gly Cys His 130 135 6 139 PRT Murinae gen. sp. tissue type embryoMOP1-MATURE 6 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys ThrPro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu AsnSer Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr ValSer Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu GlyTyr Ala Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn SerTyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val HisPhe Ile Asn Pro 85 90 95 Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr GlnLeu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn ValIle Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly CysHis 130 135 7 139 PRT Homo sapiens tissue type hippocampus HOP2-MATURE 7Ala Val Arg Pro Leu Arg Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu 1 5 1015 Pro Gln Ala Asn Arg Leu Pro Gly Ile Phe Asp Asp Val His Gly Ser 20 2530 His Gly Arg Gln Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gln 35 4045 Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala 50 5560 Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 65 7075 80 Ala Thr Asn His Ala Ile Leu Gln Ser Leu Val His Leu Met Lys Pro 8590 95 Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr100 105 110 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg LysAla 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 8139 PRT Murinae gen. sp. tissue type embyro MOP2-MATURE 8 Ala Ala ArgPro Leu Lys Arg Arg Gln Pro Lys Lys Thr Asn Glu Leu 1 5 10 15 Pro HisPro Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly His Gly Ser 20 25 30 Arg GlyArg Glu Val Cys Arg Arg His Glu Leu Tyr Val Arg Phe Arg 35 40 45 Asp LeuGly Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala 50 55 60 Tyr TyrCys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 65 70 75 80 AlaThr Asn His Ala Ile Leu Gln Ser Leu Val His Leu Met Lys Pro 85 90 95 AspVal Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 100 105 110Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His 115 120125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 9 101 PRTbovinae CBMP-2A-FX 9 Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp ValGly Trp Asn 1 5 10 15 Asp Trp Ile Val Ala Pro Pro Gly Tyr His Ala PheTyr Cys His Gly 20 25 30 Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn SerThr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser LysIle Pro Lys Ala 50 55 60 Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser MetLeu Tyr Leu Asp 65 70 75 80 Glu Asn Glu Lys Val Val Leu Lys Asn Tyr GlnAsp Met Val Val Glu 85 90 95 Gly Cys Gly Cys Arg 100 10 101 PRT Homosapiens tissue type hippocampus CMBP-2B-FX 10 Cys Arg Arg His Ser LeuTyr Val Asp Phe Ser Asp Val Gly Trp Asn 1 5 10 15 Asp Trp Ile Val AlaPro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly 20 25 30 Asp Cys Pro Phe ProLeu Ala Asp His Leu Asn Ser Thr Asn His Ala 35 40 45 Ile Val Gln Thr LeuVal Asn Ser Val Asn Ser Ser Ile Pro Lys Ala 50 55 60 Cys Cys Val Pro ThrGlu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp 65 70 75 80 Glu Tyr Asp LysVal Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu 85 90 95 Gly Cys Gly CysArg 100 11 102 PRT Drosophila melanogaster DPP-FX 11 Cys Arg Arg His SerLeu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp 1 5 10 15 Asp Trp Ile ValAla Pro Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly 20 25 30 Lys Cys Pro PhePro Leu Ala Asp His Phe Asn Ser Thr Asn His Ala 35 40 45 Val Val Gln ThrLeu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 50 55 60 Ala Cys Cys ValPro Thr Gln Leu Asp Ser Val Ala Met Leu Tyr Leu 65 70 75 80 Asn Asp GlnSer Thr Val Val Leu Lys Asn Tyr Gln Glu Met Thr Val 85 90 95 Val Gly CysGly Cys Arg 100 12 102 PRT Xenopus sp. VGL-FX 12 Cys Lys Lys Arg His LeuTyr Val Glu Phe Lys Asp Val Gly Trp Gln 1 5 10 15 Asn Trp Val Ile AlaPro Gln Gly Tyr Met Ala Asn Tyr Cys Tyr Gly 20 25 30 Glu Cys Pro Tyr ProLeu Thr Glu Ile Leu Asn Gly Ser Asn His Ala 35 40 45 Ile Leu Gln Thr LeuVal His Ser Ile Glu Pro Glu Asp Ile Pro Leu 50 55 60 Pro Cys Cys Val ProThr Lys Met Ser Pro Ile Ser Met Leu Phe Tyr 65 70 75 80 Asp Asn Asn AspAsn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 85 90 95 Asp Glu Cys GlyCys Arg 100 13 102 PRT Murinae gen. sp. VGR-1-FX 13 Cys Lys Lys His GlyLeu Tyr Val Ser Phe Gln Asp Val Gly Trp Gln 1 5 10 15 Asp Trp Ile IleAla Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 20 25 30 Glu Cys Ser PhePro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 35 40 45 Ile Val Gln ThrLeu Val His Val Met Asn Pro Glu Tyr Val Pro Lys 50 55 60 Pro Cys Cys AlaPro Thr Lys Val Asn Ala Ile Ser Val Leu Tyr Phe 65 70 75 80 Asp Asp AsnSer Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 85 90 95 Arg Ala CysGly Cys His 100 14 106 PRT Homo sapiens tissue type BRAIN GDF-1 (fx) 14Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His 1 5 1015 Arg Trp Val Ile Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gln Gly 20 2530 Gln Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala 35 4045 Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly 50 5560 Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser 65 7075 80 Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gln Tyr Glu 8590 95 Asp Met Val Val Asp Glu Cys Gly Cys Arg 100 105 15 1873 DNAMurinae gen. sp. CDS (104)..(1393) osteogenic protein MOP1, MOP1 cDNA 15ctgcagcaag tgacctcggg tcgtggaccg ctgccctgcc ccctccgctg ccacctgggg 60cggcgcgggc ccggtgcccc ggatcgcgcg tagagccggc gcg atg cac gtg cgc 115 MetHis Val Arg 1 tcg ctg cgc gct gcg gcg cca cac agc ttc gtg gcg ctc tgggcg cct 163 Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp AlaPro 5 10 15 20 ctg ttc ttg ctg cgc tcc gcc ctg gcc gat ttc agc ctg gacaac gag 211 Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp AsnGlu 25 30 35 gtg cac tcc agc ttc atc cac cgg cgc ctc cgc agc cag gag cggcgg 259 Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg Arg40 45 50 gag atg cag cgg gag atc ctg tcc atc tta ggg ttg ccc cat cgc ccg307 Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg Pro 5560 65 cgc ccg cac ctc cag gga aag cat aat tcg gcg ccc atg ttc atg ttg355 Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met Leu 7075 80 gac ctg tac aac gcc atg gcg gtg gag gag agc ggg ccg gac gga cag403 Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gln 8590 95 100 ggc ttc tcc tac ccc tac aag gcc gtc ttc agt acc cag ggc ccccct 451 Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly Pro Pro105 110 115 tta gcc agc ctg cag gac agc cat ttc ctc act gac gcc gac atggtc 499 Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp Met Val120 125 130 atg agc ttc gtc aac cta gtg gaa cat gac aaa gaa ttc ttc caccct 547 Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro135 140 145 cga tac cac cat cgg gag ttc cgg ttt gat ctt tcc aag atc cccgag 595 Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile Pro Glu150 155 160 ggc gaa cgg gtg acc gca gcc gaa ttc agg atc tat aag gac tacatc 643 Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Tyr Ile165 170 175 180 cgg gag cga ttt gac aac gag acc ttc cag atc aca gtc tatcag tgg 691 Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln Ile Thr Val Tyr GlnTrp 185 190 195 ctc cag gag cac tca ggc agg gag tcg gac ctc ttc ttg ctggac agc 739 Leu Gln Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu AspSer 200 205 210 cgc acc atc tgg gct tct gag gag ggc tgg ttg gtg ttt gatatc aca 787 Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp IleThr 215 220 225 gcc acc agc aac cac tgg gtg gtc aac cct cgg cac aac ctgggc tta 835 Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu GlyLeu 230 235 240 cag ctc tct gtg gag acc ctg gat ggg cag agc atc aac cccaag ttg 883 Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro LysLeu 245 250 255 260 gca ggc ctg att gga cgg cat gga ccc cag aac aag caaccc ttc atg 931 Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln ProPhe Met 265 270 275 gtg gcc ttc ttc aag gcc acg gaa gtc cat ctc cgt agtatc cgg tcc 979 Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser IleArg Ser 280 285 290 acg ggg ggc aag cag cgc agc cag aat cgc tcc aag acgcca aag aac 1027 Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr ProLys Asn 295 300 305 caa gag gcc ctg agg atg gcc agt gtg gca gaa aac agcagc agt gac 1075 Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser SerSer Asp 310 315 320 cag agg cag gcc tgc aag aaa cat gag ctg tac gtc agcttc cga gac 1123 Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser PheArg Asp 325 330 335 340 ctt ggc tgg cag gac tgg atc att gca cct gaa ggctat gct gcc tac 1171 Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly TyrAla Ala Tyr 345 350 355 tac tgt gag gga gag tgc gcc ttc cct ctg aac tcctac atg aac gcc 1219 Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser TyrMet Asn Ala 360 365 370 acc aac cac gcc atc gtc cag aca ctg gtt cac ttcatc aac cca gac 1267 Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe IleAsn Pro Asp 375 380 385 aca gta ccc aag ccc tgc tgt gcg ccc acc cag ctcaac gcc atc tct 1315 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu AsnAla Ile Ser 390 395 400 gtc ctc tac ttc gac gac agc tct aat gtc gac ctgaag aag tac aga 1363 Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu LysLys Tyr Arg 405 410 415 420 aac atg gtg gtc cgg gcc tgt ggc tgc cactagctcttcc tgagaccctg 1413 Asn Met Val Val Arg Ala Cys Gly Cys His 425430 acctttgcgg ggccacacct ttccaaatct tcgatgtctc accatctaag tctctcactg1473 cccaccttgg cgaggagaac agaccaacct ctcctgagcc ttccctcacc tcccaaccgg1533 aagcatgtaa gggttccaga aacctgagcg tgcagcagct gatgagcgcc ctttccttct1593 ggcacgtgac ggacaagatc ctaccagcta ccacagcaaa cgcctaagag caggaaaaat1653 gtctgccagg aaagtgtcca gtgtccacat ggcccctggc gctctgagtc tttgaggagt1713 aatcgcaagc ctcgttcagc tgcagcagaa ggaagggctt agccagggtg ggcgctggcg1773 tctgtgttga agggaaacca agcagaagcc actgtaatga tatgtcacaa taaaacccat1833 gaatgaaaaa aaaaaaaaaa aaaaaaaaaa aaaagaattc 1873 16 430 PRT Murinaegen. sp. 16 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe ValAla 1 5 10 15 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala AspPhe Ser 20 25 30 Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg LeuArg Ser 35 40 45 Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile LeuGly Leu 50 55 60 Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn SerAla Pro 65 70 75 80 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val GluGlu Ser Gly 85 90 95 Pro Asp Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala ValPhe Ser Thr 100 105 110 Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser HisPhe Leu Thr Asp 115 120 125 Ala Asp Met Val Met Ser Phe Val Asn Leu ValGlu His Asp Lys Glu 130 135 140 Phe Phe His Pro Arg Tyr His His Arg GluPhe Arg Phe Asp Leu Ser 145 150 155 160 Lys Ile Pro Glu Gly Glu Arg ValThr Ala Ala Glu Phe Arg Ile Tyr 165 170 175 Lys Asp Tyr Ile Arg Glu ArgPhe Asp Asn Glu Thr Phe Gln Ile Thr 180 185 190 Val Tyr Gln Trp Leu GlnGlu His Ser Gly Arg Glu Ser Asp Leu Phe 195 200 205 Leu Leu Asp Ser ArgThr Ile Trp Ala Ser Glu Glu Gly Trp Leu Val 210 215 220 Phe Asp Ile ThrAla Thr Ser Asn His Trp Val Val Asn Pro Arg His 225 230 235 240 Asn LeuGly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile 245 250 255 AsnPro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys 260 265 270Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 275 280285 Ser Ile Arg Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys 290295 300 Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn305 310 315 320 Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu LeuTyr Val 325 330 335 Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile AlaPro Glu Gly 340 345 350 Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala PhePro Leu Asn Ser 355 360 365 Tyr Met Asn Ala Thr Asn His Ala Ile Val GlnThr Leu Val His Phe 370 375 380 Ile Asn Pro Asp Thr Val Pro Lys Pro CysCys Ala Pro Thr Gln Leu 385 390 395 400 Asn Ala Ile Ser Val Leu Tyr PheAsp Asp Ser Ser Asn Val Asp Leu 405 410 415 Lys Lys Tyr Arg Asn Met ValVal Arg Ala Cys Gly Cys His 420 425 430

What is claimed is:
 1. A composition for increasing the progenitor cellpopulation in a mammal comprising: progenitor cells, stimulated ex vivoby exposure to a morphogen at a concentration and for a time sufficientsuch that said progenitor cells are stimulated to proliferate.
 2. Acomposition for inducing non-chondrogenic tissue growth in a mammalcomprising: progenitor cells, stimulated by exposure to a morphogen at aconcentration and for a time sufficient such that said progenitor cells,when disposed in vivo within a tissue locus, are capable ofnon-chondrogenic tissue-specific differentiation and proliferationwithin said locus.
 3. The composition of claim 1 or 2 wherein saidprogenitor cells are hemopoietic pluripotential stem cells.
 4. Thecomposition of claim 1 or 2 wherein said progenitor cells are ofmesenchymal origin.
 5. The composition of claim 1 or 2 wherein saidprogenitor cells are obtained from embryonic mesenchymal cells.
 6. Acomposition for inducing the formation of non-chondrogenic replacementtissue at a tissue locus in a mammal comprising: a biocompatible,acellular matrix having components specific for said tissue and capableof providing a morphogenically permissive, tissue-specific environment;and a morphogen such that said morphogen, when absorbed on said matrixand provided to a tissue-specific locus requiring replacement tissue, iscapable of inducing the developmental cascade of tissue morphogenesis atsaid locus.
 7. A composition for inducing the formation ofnon-chondrogenic replacement tissue at a tissue locus in a mammalcomprising: a biocompatible, acellular matrix capable of providing amorphogenically permissive environment; and a morphogen such that saidmorphogen, when absorbed on said matrix and provided to atissue-specific locus requiring replacement tissue, is capable ofinducing the developmental cascade of tissue morphogenesis at saidlocus.
 8. The composition of claim 6 or 7 wherein said matrix isbiodegradable.
 9. The composition of claim 6 or 7 wherein said matrix isderived from organ-specific tissue.
 10. The composition of claim 6 or 7wherein said matrix comprises collagen.
 11. The composition of claim 10wherein said collagen is specific for said tissue.
 12. The compositionof claim 11 further comprising cell attachment factors specific for saidtissue.
 13. The composition of claim 12 wherein said cell attachmentfactors are selected from the group consisting of glycosaminoglycans andproteoglycans.
 14. The composition of claim 6 or 7 wherein said matrixdefines pores of a dimension sufficient to permit the influx,differentiation and proliferation of migratory progenitor cells from thebody of said mammal.
 15. The composition of claim 14 wherein said matrixis treated with an agent to increase the number of pores and micropitson the matrix surface.
 16. The composition of claim 1, 2, 6 or 7 whereinsaid morphogen comprises an amino acid sequence sharing at least 70%homology with one of the sequences selected from the group consistingof: OP-1, OP-2, CMP2, Vgl(fx), Vgr(fx), DPP(fx), GDF-1(fx).
 17. Thecomposition of claim 16 wherein said morphogen comprises an amino acidsequence sharing at least 80% homology with one of the sequencesselected from said group.
 18. A method of increasing a population ofprogenitor cells comprising the step of: contacting progenitor cellswith a morphogen at a concentration and for a time sufficient such thatsaid progenitor cells are stimulated to proliferate.
 19. The method ofclaim 18 for increasing progenitor cells in a mammal comprising theadditional step of supplying said stimulated progenitor cells to amammal to increase the progenitor cell population in said mammal.
 20. Amethod of inducing non-chondrogenic tissue growth in a mammal comprisingthe step of: contacting progenitor cells with a morphogen at aconcentration and for a time sufficient such that said progenitor cells,when provided to a tissue-specific locus in a mammal, are capable ofnonchondrogenic tissue-specific differentiation and proliferation atsaid locus.
 21. The method of claim 18 or 20 wherein said progenitorcells are hemopoietic pluripotential stem cells.
 22. The method of claim18 or 20 wherein said progenitor cells are of mesenchymal origin. 23.The method of claim 18 or 20 wherein said progenitor cell are obtainedfrom embryonic mesenchymal cells.
 24. A method of maintaining thephenotypic expression of differentiated cells in a mammal comprising thesteps of: contacting said differentiated cells with a morphogen at aconcentration and for a time sufficient such that said cells arestimulated to express their phenotype.
 25. The method of claim 24wherein said differentiated cells are senescent or quiescent cells. 26.A method of inducing non-chondrogenic tissue growth at a tissue locus ina mammal comprising: providing said locus with a morphogen at aconcentration and for a time sufficient such that said protein, whenprovided to a morphogenetically permissive tissue-specific locus, iscapable of inducing the developmental cascade of tissue morphogenesis atsaid locus.
 27. The method of claim 26 wherein said nonchondrogenictissue is hepatic tissue, and said tissue locus is the liver.
 28. Themethod of claim 26 wherein said protein is provided to said locus inassociation with a biocompatible, acellular matrix.
 29. The method ofclaim 28 wherein said matrix has components specific for said tissue.30. The method of claim 28 wherein said matrix is biodegradable.
 31. Themethod of claim 28 wherein said matrix is derived from organ-specifictissue.
 32. The method of claim 28 wherein said matrix comprisescollagen and cell attachment factors specific for said tissue.
 33. Themethod of claim 32 wherein said cell attachment factors are selectedfrom the group consisting of glycosaminoglycans and proteoglycans. 34.The method of claim 28 wherein said matrix defines pores of a dimensionsufficient to permit the influx, differentiation and proliferation ofmigratory progenitor cells from the body of said mammal.
 35. The methodof claim 34 wherein said matrix is treated with an agent to increase thenumber of pores and micropits on the matrix surface.
 36. The method ofclaim 18, 20, 22 or 26 where said morphogen comprises an amino acidsequence sharing at least 70% homology with one of the sequencesselected from the group consisting of OP-1, OP-2, CBMP2, Vgl(fx),Vgr(fx), DPP(fx), and GDF-1(fx).
 37. The method of claim 36 wherein saidmorphogen comprises an amino acid sequence sharing at least 80% homologywith one of the sequences from said group.
 38. A method for inducinghepatic tissue formation at a damaged tissue locus in a mammalian livercomprising providing to said locus a therapeutic amount of a morphogencomprising at least residues 38-139 of OP-1 (Seq. ID No. 5).
 39. Amethod for detecting a pathological state in a human, the methodcomprising the step of monitoring fluctuations in physiologicalanti-morphogen antibody titer, said fluctuations being indicative ofchanges in morphogen concentrations.